The 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 fumigatus sensu lato isolates. A total of 847 A. fumigatus sensu 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.
KEYWORDS: Aspergillus fumigatus, EUCAST, azoles, amphotericin B, spectrophotometric
ABSTRACT
The 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 fumigatus sensu lato isolates. A total of 847 A. fumigatus sensu 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. fumigatus sensu lato isolates may be a convenient alternative to visual endpoint readings.
INTRODUCTION
Azoles are the backbone of treatment and prevention of diseases associated with Aspergillus spp. and are to date the only available anti-Aspergillus oral drugs. The European Society of Clinical Microbiology and Infectious Diseases guidelines recommend itraconazole for the management of patients with chronic pulmonary aspergillosis and allergic bronchopulmonary aspergillosis. Voriconazole and isavuconazole are indicated as the first-line treatment of pulmonary invasive aspergillosis. Voriconazole is also recommended for primary therapy in patients with central nervous system involvement and chronic pulmonary forms of the infection. Posaconazole is recommended for antifungal prophylaxis during prolonged neutropenia in high-risk patients or as salvage therapy in intolerant or nonresponding individuals. Finally, liposomal amphotericin B is recommended in settings in which azoles are contraindicated—resistant isolates—and as salvage therapy (1). Some Aspergillus species are intrinsically resistant to polyenes (A. terreus, A. nidulans, and A. flavus) or azoles (A. ustus) (2). Members of Aspergillus fumigatus sensu lato, the main etiological agents of aspergillosis, include A. fumigatus sensu stricto and cryptic species. Cryptic species commonly show intrinsic resistance to amphotericin B and azoles (3). In contrast, A. fumigatus sensu stricto isolates may acquire resistance following exposure to azoles, particularly with environmental azole fungicides (4). Azole resistance in A. fumigatus sensu stricto isolates has been increasingly reported worldwide (5–7).
Patients infected by azole-resistant A. fumigatus sensu lato isolates show higher mortality than those with azole-susceptible infections (8, 9). Thus, to improve patient care, detection of resistance is of paramount importance. The Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) proposed standard methods for the study of azole and amphotericin B susceptibility of Aspergillus isolates. The EUCAST EDef 9.3.2 procedure includes clinical breakpoints to classify isolates either as susceptible or resistant and recommends visual determination of MICs (10). Visual inspection may be challenging, and spectrophotometric readings may facilitate MIC determination and overcome subjectivity. However, there is a limited number of studies using the EUCAST methodology in which azole MICs against A. fumigatus sensu lato obtained by visual and spectrophotometric readings are compared; furthermore, the available studies are thwarted by a low number of isolates and antifungal drugs tested (11–14).
We recently conducted a Spanish multicenter study of azole resistance in which 847 A. fumigatus sensu lato clinical isolates were collected between 15 February and 14 May 2019 (15). Taking advantage of the large number of isolates, the objective in this work is to report and compare azole and amphotericin B MICs using visual and spectrophotometric readings following the EUCAST EDef 9.3.2 procedure.
RESULTS
Isolates classified as resistant/non-wild type according to the updated 2020 EUCAST breakpoints are listed in Table 1, as described in more detail in Materials and Methods. Tables 2 to 6 show MIC distributions of amphotericin B, itraconazole, posaconazole, voriconazole, and isavuconazole against the 847 isolates by regular/stringent visual and spectrophotometric readings. MICs against quality control (QC) strains were within the acceptable limit.
TABLE 1.
Azole and amphotericin B breakpoints and ECOFFs chosen to classify Aspergillus fumigatus sensu lato isolates as susceptible, resistant, or non-wild typea
| Drug | ECOFF for WT (mg/liter) | Clinical breakpoint (mg/liter) |
||
|---|---|---|---|---|
| S | R | ATUb | ||
| Amphotericin B | ≤1 | ≤1 | ≥2 | ND |
| Itraconazole | ≤1 | ≤1 | ≥2 | 2 |
| Posaconazole | ≤0.25 | ≤0.125 | ≥0.25 | 0.25 |
| Voriconazole | ≤1 | ≤1 | ≥2 | 2 |
| Isavuconazole | ≤2 | ≤1 | ≥4 | 2 |
For details, see reference 17. ECOFF, epidemiological cutoff value; WT, wild type; S, susceptible; R, resistant; ATU, area of technical uncertainty; ND, not defined.
Isolates with itraconazole and voriconazole MIC results that fall in the ATU were always considered resistant; isolates with isavuconazole MICs and posaconazole MIC results that fall in the ATU were considered resistant when voriconazole resistant or itraconazole resistant, respectively.
TABLE 2.
MIC distributions of amphotericin B against 847 A. fumigatus sensu lato isolatesa
| MIC reading procedure | MIC distribution by no. of isolates for MIC (mg/liter) of: |
No. (%) of resistant isolatesb | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 0.008 | 0.016 | 0.03 | 0.06 | 0.125 | 0.25 | 0.5 | 1 | 2 | 4 | 8 | ≥16 | ||
| A. fumigatus sensu lato (n = 847) | |||||||||||||
| Regular visual readings | 0 | 0 | 1 | 5 | 70 | 407 | 306 | 45 | 7 | 5 | 1 | 0 | 13 (1.5) |
| Spectrophotometric readings | 0 | 0 | 0 | 7 | 96 | 452 | 248 | 31 | 6 | 5 | 0 | 2 | 13 (1.5) |
| A. fumigatus sensu stricto (n = 828) | |||||||||||||
| Regular visual readings | 0 | 0 | 1 | 5 | 68 | 407 | 305 | 42 | 0 | 0 | 0 | 0 | 0 (0) |
| Spectrophotometric readings | 0 | 0 | 0 | 7 | 94 | 452 | 246 | 29 | 0 | 0 | 0 | 0 | 0 (0) |
| Cryptic species (n = 19) | |||||||||||||
| Regular visual readings | 0 | 0 | 0 | 0 | 2 | 0 | 1 | 3 | 7 | 5 | 1 | 0 | 13 (68.4) |
| Spectrophotometric readings | 0 | 0 | 0 | 0 | 2 | 0 | 2 | 2 | 6 | 5 | 0 | 2 | 13 (68.4) |
| Isolates with tandem repeats (n = 25) | |||||||||||||
| Regular visual readings | 0 | 0 | 0 | 0 | 2 | 10 | 13 | 0 | 0 | 0 | 0 | 0 | 0 (0) |
| Spectrophotometric readings | 0 | 0 | 0 | 0 | 3 | 10 | 12 | 0 | 0 | 0 | 0 | 0 | 0 (0) |
MIC distributions by regular visual readings and their correspondent rates of resistance are reported elsewhere (15). Underlined values indicate non-wild-type isolates according to tentative ECOFFs, and values in boldface indicate resistant isolates (EUCAST breakpoint table v. 10.0, 2020 [10]).
Identical numbers of resistant isolates and non-wild-type isolates were obtained.
TABLE 3.
MIC distributions of itraconazole against 847 A. fumigatus sensu lato isolatesa
| MIC reading procedure | MIC distribution by no. of isolates for MIC (mg/liter) of: |
No. (%) of resistant isolatesb | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 0.008 | 0.016 | 0.03 | 0.06 | 0.125 | 0.25 | 0.5 | 1 | 2 | 4 | 8 | ≥16 | ||
| A. fumigatus sensu lato (n = 847) | |||||||||||||
| Regular visual readings | 0 | 0 | 0 | 0 | 26 | 427 | 328 | 21 | 2 | 2 | 2 | 39 | 45 (5.3) |
| Stringent visual readings | 0 | 0 | 0 | 0 | 10 | 269 | 459 | 59 | 2 | 2 | 2 | 44 | 50 (5.9) |
| Spectrophotometric readings | 0 | 0 | 15 | 22 | 49 | 412 | 287 | 17 | 3 | 1 | 1 | 40 | 45 (5.3) |
| A. fumigatus sensu stricto (n = 828) | |||||||||||||
| Regular visual readings | 0 | 0 | 0 | 0 | 26 | 426 | 326 | 15 | 2 | 1 | 1 | 31 | 35 (4.2) |
| Stringent visual readings | 0 | 0 | 0 | 0 | 10 | 269 | 457 | 57 | 2 | 1 | 1 | 31 | 35 (4.2) |
| Spectrophotometric readings | 0 | 0 | 15 | 22 | 49 | 410 | 282 | 15 | 3 | 0 | 1 | 31 | 35 (4.2) |
| Cryptic species (n = 19) | |||||||||||||
| Regular visual readings | 0 | 0 | 0 | 0 | 0 | 1 | 2 | 6 | 0 | 1 | 1 | 8 | 10 (52.6) |
| Stringent visual readings | 0 | 0 | 0 | 0 | 0 | 0 | 2 | 2 | 0 | 1 | 1 | 13 | 15 (78.9) |
| Spectrophotometric readings | 0 | 0 | 0 | 0 | 0 | 2 | 5 | 2 | 0 | 1 | 0 | 9 | 10 (52.6) |
| Isolates with tandem repeats (n = 25) | |||||||||||||
| Regular visual readings | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 24 | 24 (96) |
| Stringent visual readings | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 24 | 24 (96) |
| Spectrophotometric readings | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 24 | 24 (96) |
MIC distributions by regular visual readings and their correspondent rates of resistance are reported elsewhere (15). Values shaded in gray indicate MICs in the area of technical uncertainty (ATU) and were classified as resistant isolates. Underlined values indicate non-wild-type isolates according to tentative ECOFFs, and values in boldface indicate resistant isolates (EUCAST breakpoint table v. 10.0, 2020 [10]).
Identical numbers of resistant isolates and non-wild-type isolates were obtained.
TABLE 4.
MIC distributions of posaconazole against 847 A. fumigatus sensu lato isolatesa
| MIC reading procedure | MIC distribution by no. of isolates for MIC (mg/liter) of: |
No. (%) of isolates |
||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 0.008 | 0.016 | 0.03 | 0.06 | 0.125 | 0.25 | 0.5 | 1 | 2 | 4 | 8 | ≥16 | Resistant | Non-wild type | |
| A. fumigatus sensu lato (n = 847) | ||||||||||||||
| Regular visual readings | 0 | 2 | 46 | 441 | 279 | 42 | 27 | 3 | 0 | 1 | 0 | 6 | 46 (5.4) | 37 (4.4) |
| Stringent visual readings | 0 | 1 | 18 | 268 | 399 | 119 | 23 | 12 | 0 | 1 | 0 | 6 | 51 (6) | 42 (5) |
| Spectrophotometric readings | 1 | 1 | 70 | 476 | 225 | 36 | 28 | 4 | 1 | 1 | 0 | 4 | 47 (5.5) | 38 (4.5) |
| A. fumigatus sensu stricto (n = 828) | ||||||||||||||
| Regular visual readings | 0 | 2 | 46 | 440 | 278 | 32 | 20 | 3 | 0 | 1 | 0 | 6 | 34 (4.1) | 30 (3.6) |
| Stringent visual readings | 0 | 1 | 18 | 268 | 398 | 111 | 13 | 12 | 0 | 1 | 0 | 6 | 34 (4.1) | 32 (3.9) |
| Spectrophotometric readings | 1 | 1 | 70 | 475 | 222 | 27 | 22 | 4 | 1 | 1 | 0 | 4 | 34 (4.1) | 32 (3.9) |
| Cryptic species (n = 19) | ||||||||||||||
| Regular visual readings | 0 | 0 | 0 | 1 | 1 | 10 | 7 | 0 | 0 | 0 | 0 | 0 | 12 (63.1) | 7 (36.8) |
| Stringent visual readings | 0 | 0 | 0 | 0 | 1 | 8 | 10 | 0 | 0 | 0 | 0 | 0 | 17 (89.5) | 10 (52.6) |
| Spectrophotometric readings | 0 | 0 | 0 | 1 | 3 | 9 | 6 | 0 | 0 | 0 | 0 | 0 | 12 (63.1) | 6 (31.6) |
| Isolates with tandem repeats (n = 25) | ||||||||||||||
| Regular visual readings | 0 | 0 | 0 | 0 | 0 | 3 | 18 | 2 | 0 | 0 | 0 | 2 | 24 (96) | 22 (88) |
| Stringent visual readings | 0 | 0 | 0 | 0 | 0 | 1 | 11 | 11 | 0 | 0 | 0 | 2 | 24 (96) | 24 (96) |
| Spectrophotometric readings | 0 | 0 | 0 | 0 | 0 | 1 | 19 | 4 | 1 | 0 | 0 | 0 | 24 (96) | 24 (96) |
MIC distributions by regular visual readings and their correspondent rates of resistance or non-wild-type isolates are reported elsewhere (15). Values shaded in gray indicate MICs in the area of technical uncertainty (ATU). MIC results against A. fumigatus sensu lato falling in the ATU were translated to resistant as follows: regular visual readings (n = 9/42), stringent visual readings (n = 9/119), and spectrophotometric readings (n = 9/36). Underlined values indicate non-wild-type isolates according to tentative ECOFFs, and values in boldface indicate resistant isolates (EUCAST Breakpoint table v. 10.0, 2020 [10]).
TABLE 5.
MIC distributions of voriconazole against 847 A. fumigatus sensu lato isolatesa
| MIC reading procedure | MIC distribution by no. of isolates for MIC (mg/liter) of: |
No. (%) of resistant isolatesb | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 0.008 | 0.016 | 0.03 | 0.06 | 0.125 | 0.25 | 0.5 | 1 | 2 | 4 | 8 | ≥16 | ||
| A. fumigatus sensu lato (n = 847) | |||||||||||||
| Visual reading | 0 | 0 | 0 | 0 | 3 | 82 | 529 | 177 | 19 | 27 | 7 | 3 | 56 (6.6) |
| Stringent visual readings | 0 | 0 | 0 | 0 | 1 | 17 | 302 | 394 | 87 | 31 | 11 | 4 | 133 (15.7) |
| Spectrophotometric readings | 0 | 0 | 0 | 0 | 4 | 138 | 500 | 154 | 18 | 24 | 6 | 3 | 51 (6) |
| A. fumigatus sensu stricto (n = 828) | |||||||||||||
| Visual reading | 0 | 0 | 0 | 0 | 3 | 82 | 529 | 176 | 13 | 19 | 3 | 3 | 38 (4.6) |
| Stringent visual readings | 0 | 0 | 0 | 0 | 1 | 17 | 302 | 393 | 86 | 18 | 7 | 4 | 115 (13.9) |
| Spectrophotometric readings | 0 | 0 | 0 | 0 | 4 | 138 | 498 | 153 | 11 | 18 | 4 | 2 | 35 (4.2) |
| Cryptic species (n = 19) | |||||||||||||
| Visual reading | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 6 | 8 | 4 | 0 | 18 (94.7) |
| Stringent visual readings | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 1 | 13 | 4 | 0 | 18 (94.7) |
| Spectrophotometric readings | 0 | 0 | 0 | 0 | 0 | 0 | 2 | 1 | 7 | 6 | 2 | 1 | 16 (84.2) |
| Isolates with tandem repeats (n = 25) | |||||||||||||
| Visual reading | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 5 | 15 | 2 | 3 | 25 (100) |
| Stringent visual readings | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 14 | 7 | 3 | 25 (100) |
| Spectrophotometric readings | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 6 | 14 | 3 | 2 | 25 (100) |
MIC distributions by regular visual readings and their correspondent rates of resistance are reported elsewhere (15). Values shaded in gray indicate MICs in the area of technical uncertainty (ATU) and were classified as resistant isolates. Underlined values indicate non-wild-type isolates according to tentative ECOFFs, and values in boldface indicate resistant isolates (EUCAST breakpoint table v. 10.0, 2020 [10]).
The numbers of resistant isolates and non-wild-type isolates were identical.
TABLE 6.
MIC distributions of isavuconazole against 847 A. fumigatus sensu lato isolatesa
| MIC reading procedure | MIC distribution by no. of isolates for MIC (mg/liter) of: |
No. of isolates (%) |
||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 0.008 | 0.016 | 0.03 | 0.06 | 0.125 | 0.25 | 0.5 | 1 | 2 | 4 | 8 | ≥16 | Resistant | Non-wild type | |
| A. fumigatus sensu lato (n = 847) | ||||||||||||||
| Regular visual readings | 0 | 0 | 0 | 0 | 0 | 13 | 440 | 333 | 26 | 14 | 17 | 4 | 48 (5.6) | 35 (4.1) |
| Stringent visual readings | 0 | 0 | 0 | 0 | 0 | 2 | 146 | 572 | 90 | 11 | 21 | 5 | 72 (8.5) | 37 (4.4) |
| Spectrophotometric readings | 0 | 0 | 0 | 17 | 4 | 14 | 434 | 314 | 31 | 12 | 18 | 3 | 42 (5) | 33 (3.9) |
| A. fumigatus sensu stricto (n = 828) | ||||||||||||||
| Regular visual readings | 0 | 0 | 0 | 0 | 0 | 13 | 440 | 327 | 18 | 10 | 16 | 4 | 35 (4.2) | 30 (3.6) |
| Stringent visual readings | 0 | 0 | 0 | 0 | 0 | 2 | 146 | 568 | 80 | 7 | 20 | 5 | 57 (6.9) | 32 (3.9) |
| Spectrophotometric readings | 0 | 0 | 0 | 17 | 4 | 14 | 433 | 306 | 24 | 9 | 18 | 3 | 32 (3.9) | 30 (3.6) |
| Cryptic species (n = 19) | ||||||||||||||
| Regular visual readings | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 6 | 8 | 4 | 1 | 0 | 13 (68.4) | 5 (26.3) |
| Stringent visual readings | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 4 | 10 | 4 | 1 | 0 | 15 (78.9) | 5 (26.3) |
| Spectrophotometric readings | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 8 | 7 | 3 | 0 | 0 | 10 (52.6) | 3 (15.8) |
| Isolates with tandem repeats (n = 25) | ||||||||||||||
| Regular visual readings | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 6 | 14 | 4 | 25 (100) | 24 (96) |
| Stringent visual readings | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 2 | 18 | 5 | 25 (100) | 25 (100) |
| Spectrophotometric readings | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 7 | 15 | 3 | 25 (100) | 25 (100) |
MIC distributions by regular visual readings and their correspondent rates of resistance/non-wild-type isolates were reported elsewhere (15). Values shaded in gray indicate MICs in the area of technical uncertainty (ATU). MIC results against A. fumigatus sensu lato falling in the ATU were translated to resistant as follows: regular visual readings (n = 13/26), stringent visual readings (n = 35/90), and spectrophotometric readings (n = 9/31). Underlined values indicate non-wild-type isolates according to tentative ECOFFs, and values in boldface indicate resistant isolates (EUCAST breakpoint table v. 10.0, 2020 [10]).
Agreement between MICs by regular visual and spectrophotometric readings.
Overall, both MIC endpoints showed high essential (97.1%) and categorical (99.6%) agreements. Essential agreements for individual drugs were as follows: amphotericin B, 98.8%; itraconazole, 94.8%; posaconazole, 97.3%; voriconazole, 98.3%; and isavuconazole, 96.1% (Table 7).
TABLE 7.
Essential and categorical agreement between MICs by visual (regular and stringent) and spectrophotometric readingsa
| MIC reading comparison |
A. fumigatus
sensu lato (%) |
A. fumigatus
sensu stricto (%) |
Cryptic species (%) |
|||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Essential agreement | Categorical agreement | VME | ME | Essential agreement | Categorical agreement | VME | ME | Essential agreement | Categorical agreement | VME | ME | |
| Amphotericin B | ||||||||||||
| Regular visual vs spectrophotometric readings | 98.8 | 100 | 0 | 0 | 99 | 100 | 0 | 0 | 89.5 | 100 | 0 | 0 |
| Itraconazole | ||||||||||||
| Regular visual vs spectrophotometric readings | 94.8 | 100 | 0 | 0 | 95.1 | 100 | 0 | 0 | 78.9 | 100 | 0 | 0 |
| Regular visual vs stringent readings | 98.9 | 99.4 | 0 | 0.6 | 99.6 | 100 | 0 | 0 | 68.4 | 73.7 | 0 | 26.3 |
| Posaconazole | ||||||||||||
| Regular visual vs spectrophotometric readings | 97.3 | 100 | 0 | 0 | 97.1 | 100 | 0 | 0 | 100 | 100 | 0 | 0 |
| Regular visual vs stringent readings | 98.7 | 99.4 | 0 | 0.6 | 98.7 | 100 | 0 | 0 | 100 | 73.7 | 0 | 26.3 |
| Voriconazole | ||||||||||||
| Regular visual vs spectrophotometric readings | 98.3 | 98.7 | 0.9 | 0.3 | 98.4 | 98.9 | 0.7 | 0.4 | 94.7 | 89.5 | 10.5 | 0 |
| Regular visual vs stringent readings | 94.4 | 90.8 | 0 | 9.2 | 94.3 | 90.6 | 0 | 9.4 | 100 | 100 | 0 | 0 |
| Isavuconazole | ||||||||||||
| Regular visual vs spectrophotometric readings | 96.1 | 99.3 | 0.7 | 0 | 96.3 | 99.6 | 0.4 | 0 | 89.5 | 84.2 | 15.8 | 0 |
| Regular visual vs stringent readings | 98.6 | 97.2 | 0 | 2.8 | 98.5 | 97.3 | 0 | 2.7 | 100 | 89.5 | 0 | 10.5 |
Regular visual endpoint MICs were assumed as the gold standards and compared against MICs obtained by other endpoints. MICs (percentages) within ±1 2-fold dilution were considered to be in essential agreement. Isolates were classified as resistant/non-wild type according to the updated 2020 EUCAST breakpoints/ECOFFs. The endpoints were in categorical agreement when the results were in the same susceptibility category (regardless of the MIC). VME, very major error (false susceptibility); ME, major error (false resistance).
Categorical agreements for amphotericin B, itraconazole, and posaconazole were 100%, and consequently, resistance rates for both MIC endpoints were identical. Categorical agreement for voriconazole was 98.7%, and the rate of resistance was slightly lower when spectrophotometric readings were used for MIC determination. Very major errors (0.7% [n = 6]) and major errors (0.4% [n = 3]) for voriconazole occurred in A. fumigatus sensu stricto isolates with MIC results falling in the area of technical uncertainty (ATU) (MIC = 2 mg/liter). In cryptic species, very major errors occurred in two Neosartorya udagawae isolates (10.5%), one of them with MIC results falling in the ATU. Categorical agreement for isavuconazole was 99.3%, and the rate of resistance was slightly lower when spectrophotometric readings were used for MIC determination. Very major errors in isavuconazole occurred in three A. fumigatus sensu stricto isolates and in three cryptic species isolates (two of N. udagawae and one of A. fumigatiaffinis). With the exception of the A. fumigatiaffinis isolate, very major errors for isavuconazole (n = 5) were detected in isolates with MIC results in the ATU, which also revealed very major errors for voriconazole (see Table S1 in the supplemental material). None of the six isolates for which very major errors were detected in the azole categorical classification harbored relevant cyp51A mutations (Table S1).
Agreement between MICs obtained by regular/stringent visual readings.
Overall, both visual MIC endpoints showed high essential (97.7%) and categorical (96.7%) agreements. Essential agreements for individual drugs were above 98% (itraconazole, 98.9%; posaconazole, 98.7%; and isavuconazole, 98.6%), with the exception of voriconazole (94.4%) (Table 7).
Categorical agreements for itraconazole and posaconazole were 99.4% (Table 7). Resistance rates obtained by both MIC endpoints were identical in A. fumigatus sensu stricto, but slightly higher with stringent visual readings in cryptic species. This led to major errors for both drugs in five isolates (three of A. lentulus, one of A. novofumigatus, and one of A. fumigatiaffinis). Although posaconazole MICs by both visual readings were identical (MIC = 0.25 mg/liter [ATU]), the categorical classification differed due to the MICs of itraconazole in four out of the five isolates (see Table S2 in the supplemental material). The percentage of voriconazole resistance was overestimated with the stringent visual endpoint (6.6% versus 15.8%). Categorical agreement was 90.8%. Major errors were found exclusively in A. fumigatus sensu stricto isolates (n = 78), in MIC results falling in the ATU. Likewise, the rate of isavuconazole resistance was overestimated when the stringent visual endpoint was used, although to a lesser extent than in the case of voriconazole (4.1% versus 4.4%). Categorical agreement was 97.2%. Major errors were found in A. fumigatus sensu stricto isolates (n = 22) and in two isolates of cryptic species (N. tsurutae and A. fumigatiaffinis [Table S2]). Similarly, most misclassifications (23/24 isolates) were associated with MIC results falling in the ATU and mostly affected isolates in which major errors for voriconazole were detected (21/24 isolates). Since stringent visual readings shifted azole MICs to higher values, no very major errors were found.
DISCUSSION
In this study, we show that MICs of azoles and amphotericin B against A. fumigatus obtained either by spectrophotometric or regular visual readings have very high essential and categorical agreement.
The increase in resistant A. fumigatus isolates worldwide has promoted antifungal susceptibility testing (5). Azole resistance in A. fumigatus may occur during azole therapy or exposure to azole fungicides in the environment (4). Furthermore, cryptic species commonly show intrinsic resistance to amphotericin B and azoles (3). Although the EUCAST EDef 9.3.2 procedure recommends visual inspection for azole and amphotericin B MIC settings against Aspergillus species, spectrophotometric readings may offer objectivity, quick automated readings, and overall better performance. Previous studies comparing spectrophotometric and visual readings showed excellent essential (92 to 97%) and categorical (93 to 99%) agreements (11–14). Some of the studies used the CLSI methodology and were undermined by the limited number of A. fumigatus sensu stricto isolates tested (up to 133 isolates), the absence of both cryptic species isolates and cyp51A mutants, and a low number of studied antifungal drugs (amphotericin B and itraconazole) (12–14). One of the studies, in which the EUCAST method was used, included the four antimold triazoles (itraconazole, posaconazole, voriconazole, and isavuconazole) and a low number of A. fumigatus sensu stricto isolates (n = 88). The work did not assess cryptic species, although 15 isolates with cyp51A mutations, including isolates with the dominant substitutions TR34 L98H, G54, and M220, among others, were examined. Furthermore, since EUCAST has recently changed azole breakpoints against Aspergillus fumigatus sensu lato, a validation of spectrophotometric readings, including a large number of isolates classified according to the updated EUCAST breakpoints, is needed.
We recently conducted a survey of azole resistance in A. fumigatus sensu lato isolates collected in Spain in 2019 (15). Taking advantage of the large number of isolates collected (n = 847), we obtained and compared MICs using visual and spectrophotometric readings. Nineteen strains were identified as cryptic species, and 45 A. fumigatus sensu stricto isolates proved to be azole resistant, being TR34 L98H, the dominant mechanism of resistance. Both MIC endpoints show high essential/categorical agreements for amphotericin B (98.8%/100%), itraconazole (94.8%/100%), posaconazole (97.3%/100%), voriconazole (98.3%/98.7%), and isavuconazole (96.1%/99.3%). No errors were found in amphotericin B, itraconazole, and posaconazole. Most misclassifications for voriconazole and isavuconazole are linked with MIC results falling either in the ATU (10/12 isolates) or in just one 2-fold dilution above the breakpoint (2/12 isolates; MIC = 4 mg/liter). Cross-resistance between voriconazole and isavuconazole is the norm in A. fumigatus senso stricto (16). Using voriconazole as a surrogate marker, spectrophotometric readings resulted in misdetection of voriconazole resistance in six A. fumigatus senso stricto isolates with either a wild-type cyp51A gene or genetic polymorphisms of dubious clinical implications (Table S1).
The EUCAST has recently reviewed the antifungal breakpoints against A. fumigatus sensu lato. Breakpoints for amphotericin B, itraconazole, voriconazole, and posaconazole were lowered, while the breakpoint for isavuconazole was increased (17). Based on the updated breakpoints, spectrophotometric MIC readings led to correct classification of all isolates with relevant cyp51A mutations as resistant. Interpretation uncertainties regarding MIC values may arise in the ATU, a newly introduced term, where the breakpoints of wild-type isolates and mutant isolates converge (17). Isolates with posaconazole and isavuconazole MICs of 0.25 mg/liter and 2 mg/liter, respectively, cannot be automatically reported as susceptible or resistant. MIC determinations using spectrophotometric readings frequently led to the underestimation of resistance for MIC values falling in the ATU. Here, we were able to easily clarify misclassifications by visually inspecting the tray. False resistance was detected in four A. fumigatus sensu stricto isolates for which a voriconazole MIC of 2 mg/liter was determined by spectrophotometric readings.
A higher mortality rate is observed in patients infected with azole-resistant A. fumigatus sensu lato isolates. Resistance is frequently caused by mutations in the cyp51A gene, some of which are associated with a pan-triazole-resistant phenotype (high-level resistance) (8). Some phenotypes only affect the activity of a single azole or several triazoles with similar molecular structure, and the MIC is close to the clinical breakpoint, resulting in low-level resistance (18). Previous studies have shown that patients infected with low-level voriconazole-resistant A. fumigatus (MIC = 2 mg) and low-level isavuconazole-resistant A. fumigatus (MIC = 2 mg) may be treated with voriconazole or isavuconazole, respectively, provided that higher doses are administered (19, 20). In cryptic species, very major errors in voriconazole and isavuconazole were detected (Table S1).
Visual MIC readings may be challenging, and taking small colonies into account (stringent visual readings) may result in overestimation of resistance rates and increase the MIC of the isolates one or two 2-fold dilutions, particularly for voriconazole. Thus, major errors in voriconazole and isavuconazole (MIC results falling in the ATU) against A. fumigatus sensu stricto may be detected. Correct classification of relevant cyp51A gene mutants was achieved by stringent visual readings.
We conclude that spectrophotometric determination is a useful alternative to visual inspection of azole and amphotericin MICs against A. fumigatus sensu stricto. Both endpoints show high essential and categorical agreements. Future studies that include more isolates from cryptic species and A. fumigatus sensu stricto with other kinds of cyp51A mutations are warranted.
MATERIALS AND METHODS
Samples.
A total of 847 A. fumigatus sensu lato clinical isolates, identified by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS), were collected in a 30-hospital survey conducted in Spain (15). Azole-resistant isolates (A. fumigatus sensu stricto, n = 45; cryptic species, n = 19) were molecularly identified. The distribution of isolates as per species identification was as follows: A. fumigatus sensu stricto, n = 828; A. lentulus, n = 6; A. fumigatiaffinis, n = 5; Neosartorya tsurutae, n = 3; Neosartorya udagawae, n = 2; A. novofumigatus, n = 2; and A. thermomutatus, n = 1.
The cyp51A gene sequence from 45 azole-resistant A. fumigatus sensu stricto isolates carried the mutations TR34 L98H (n = 24), G54R (n = 5), TR46 Y121F T289A (n = 1), F46Y M172V N248T D255E E427K (n = 2), F46Y M172V N248T D255E E416Q E427K (n = 1), F165L (n = 1), and S496L (n = 1), as well as the wild-type cyp51A gene (n = 10).
EUCAST antifungal susceptibility testing.
All isolates were subcultured on potato dextrose agar or Sabouraud dextrose agar and incubated at 35°C for 2 to 5 days. Isolates from cryptic species were incubated long enough to ensure filtered conidial suspensions reached a sufficient inoculum (equivalent to a McFarland standard of 0.5 using a spectrophotometer). The isolates’ antifungal susceptibilities to amphotericin B, itraconazole, voriconazole, posaconazole, and isavuconazole were determined following the EUCAST EDef 9.3.2 procedure (21). The inoculated trays were incubated for 48 h at 35°C, and MICs were obtained using a visual endpoint (defined as the concentration that completely inhibits fungal growth) and a spectrophotometric endpoint (≥95% inhibition of fungal growth compared to the drug-free control and read at 540 nm, as described elsewhere) (11). Although the EUCAST EDef 9.3.2 procedure recommends ignoring single colonies on the surface, sometimes it is difficult to discern real growth from small colonies. Thus, we interpreted visual MICs using two endpoints—the regular endpoint (very tiny growth was disregarded) or the stringent endpoint (a totally clear well)—as exemplified in Fig. 1. Quality control (QC) was ensured by testing the A. flavus ATCC 204304 and A. fumigatus ATCC 204305 strains (amphotericin B, itraconazole, voriconazole, and posaconazole), as well as Candida krusei ATCC 6258 and Candida parapsilosis ATCC 22019 (isavuconazole).
FIG 1.
Example of fungal growth of an A. fumigatus sensu stricto isolate in the presence of itraconazole (a), posaconazole (b), voriconazole (c), and isavuconazole (d). Two MIC endpoints were used: the regular endpoint (gold standard), where tiny small colonies were disregarded (wells surrounded by rings of dashed lines), and the stringent endpoint, where the tiny colonies were taken into account (wells surrounded by rings of solid lines).
Data analysis.
Regular visual endpoint MICs were assumed as the “gold standards” and compared against MICs obtained by other endpoints; MICs (percentage) within ±1 2-fold dilution were considered to be in essential agreement. Isolates were classified as resistant/non-wild type according to the updated 2020 EUCAST breakpoints (Table 1); the intermediate category for amphotericin B and azoles and the category of “susceptible increased exposure” are no longer available, and the term “area of technical uncertainty” (ATU) for the four azoles has been defined (10). The ATU is a warning to laboratories on an uncertainty needing attention before reporting the results and represents an area of confluence of both wild-type and mutant isolates, particularly for voriconazole, posaconazole, and isavuconazole. MIC results in the ATU were interpreted as follows: itraconazole and voriconazole, always resistant; posaconazole, resistant only if the isolate was also resistant to itraconazole; and isavuconazole, resistant only if the isolate was also resistant to voriconazole. Categorical agreement between the three endpoints was assessed. The endpoints were in categorical agreement when the results were in the same susceptibility category (regardless of the MIC). Errors were defined as very major errors (false susceptibility) when the gold standard endpoint classified an isolate as resistant and the other endpoints as susceptible and as major errors (false resistance) when the gold standard endpoint classified an isolate as susceptible and the other endpoints as resistant (22).
Ethical considerations.
This study was approved by the Ethics Committee of Hospital Gregorio Marañón (CEIm; study no. 22/19).
Supplementary Material
ACKNOWLEDGMENTS
The authors are grateful to Dainora Jaloveckas for editing assistance.
This study was supported by grants CP15/00115 from Fondo de Investigación Sanitaria (FIS; Instituto de Salud Carlos III, Plan Nacional de I+D+I 2013–2016). The study was cofunded by the European Regional Development Fund (FEDER) “A Way of Making Europe.” This work was supported by grants from Basilea Pharmaceutica, Ltd. (Basel, Switzerland). The funders had no role in the study design, data collection, analysis, decision to publish, or preparation/content of the manuscript.
P.E. (CPI15/00115) is a recipient of a Miguel Servet contract supported by the FIS. J.G. is a stabilized researcher contracted by Fundación para Investigación Sanitaria del Hospital Gregorio Marañón.
The members of the ASPEIN Study Group and their affiliations are as follows: Waldo Sánchez-Yebra and Juan Sánchez-Gómez, Complejo Hospitalario Torrecárdenas, Almería; Inmaculada Lozano, Hospital Universitario Puerta del Mar, Cádiz; Eduardo Marfil, Montserrat Muñoz de la Rosa, and Rocío Tejero García, Hospital Universitario Reina Sofía, Córdoba; Fernando Cobo, Hospital Virgen de las Nieves, Granada; Carmen Castro, Hospital de Valme, Seville; Concepción López and Antonio Rezusta, Hospital Universitario Miguel Servet, Zaragoza; Teresa Peláez, Cristian Castelló-Abietar, and Isabel Costales, Hospital Universitario Central de Asturias, Oviedo; Julia Lozano Serra, Hospital General de Albacete, Albacete; Rosa Jiménez, Complejo Hospitalario de Toledo, Toledo; Cristina Labayru Echeverría, Cristina Losa Pérez, and Gregoria Megías-Lobón, Hospital Universitario de Burgos, Burgos; Belén Lorenzo, Hospital Río Hortega, Valladolid; Ferrán Sánchez-Reus, Hospital Santa Creu i Sant Pau, Barcelona; Josefina Ayats, Hospital de Bellvitge, Barcelona; María Teresa Martín, Hospital Vall de Hebrón, Barcelona; Inmaculada Vidal, Hospital General de Alicante, Alicante; Victoria Sánchez-Hellín, Hospital General de Elche, Elche; Elisa Ibáñez and Javier Pemán, Hospital Universitario la Fe, Valencia; Miguel Fajardo, Hospital Universitario de Badajoz, Badajoz; Carmen Pazos, Hospital San Pedro de Alcántara, Cáceres; María Rodríguez-Mayo, Complejo Hospitalario Universitario de A Coruña, A Coruña; Ana Pérez-Ayala, Hospital 12 de Octubre, Madrid; Elia Gómez, Hospital Ramón y Cajal, Madrid; Jesús Guinea, Pilar Escribano, Julia Serrano, Elena Reigadas, Belén Rodríguez, Estreya Zvezdanova, Judith Díaz-García, Ana Gómez-Núñez, José González Leiva, Marina Machado, and Patricia Muñoz, Hospital General Universitario Gregorio Marañón, Madrid; Isabel Sánchez-Romero, Hospital Puerta de Hierro, Madrid; Julio García-Rodríguez, Hospital La Paz, Madrid; José Luis del Pozo and Manuel Rubio Vallejo, Clínica Universidad de Navarra, Pamplona; Carlos Ruiz de Alegría-Puig, Hospital de Valdecilla, Santander; Leyre López-Soria, Hospital de Cruces, Bilbao; José María Marimón and Diego Vicente, Hospital de Donostia, Donostia; and Marina Fernández-Torres and Silvia Hernáez-Crespo, Hospital de Txagorritxu, Vitoria-Gasteiz.
Julia Serrano-Lobo contributed formal analysis, data collection, writing—original draft preparation, review, and editing; Ana Gómez contributed experimental part, formal analysis, data collection, and supervision; Waldo Sánchez-Yebra, Miguel Fajardo, Belén Lorenzo, Ferrán Sánchez-Reus, Inmaculada Vidal, Marina Fernández-Torres, Isabel Sánchez-Romero, Carlos Ruiz de Alegría-Puig, and José Luis del Pozo contributed submission of isolates, original draft preparation, review, and editing; Patricia Muñoz contributed writing, review, and editing; Pilar Escribano contributed conceptualization, experimental part, formal analysis, data collection, supervision, validation, visualization, writing—original draft preparation, review, and editing; Jesús Guinea contributed conceptualization, project administration, formal analysis, supervision, validation, visualization, original draft preparation, review, and editing.
Footnotes
Supplemental material is available online only.
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