Species Distribution and Antifungal Susceptibilities of Aspergillus Section Fumigati Isolates in Clinical Samples from the United States

ABSTRACT

Aspergillus species are capable of causing both invasive disease and chronic infections in immunocompromised patients or those with preexisting lung conditions. Aspergillus fumigatus is the most commonly cultured species, and there is increasing concern regarding resistance to the azoles, which are the mainstays of antifungal therapy against aspergillosis. We evaluated the species distribution and susceptibility profiles of isolates within Aspergillus section Fumigati in the United States over a 52-month period. Species identification was performed by combined phenotypic characteristics and DNA sequence analysis, and antifungal susceptibility testing was performed by CLSI M38 broth microdilution for amphotericin B, the azoles, and the echinocandins. The entire CYP51A gene and its promoter were also sequenced in isolates that were phenotypically resistant to the azoles. During the study time frame, 2,138 isolates were included, representing 11 different species within Aspergillus section Fumigati, of which A. fumigatus was the most prevalent (96.91%). Overall, amphotericin B and the echinocandins demonstrated consistent in vitro activity with very few isolates demonstrating reduced susceptibility to these agents. Voriconazole, isavuconazole, and posaconazole also demonstrated good in vitro activity, and the overall percentages of isolates classified as resistant or non-wild type ranged from 3.33 to 6.58%. Mutations within the CYP51A gene leading to amino acid changes associated with azole resistance were found in 75.3% of isolates that were phenotypically resistant or non-wild type and included both those associated with chronic clinical exposure and environmental exposure to the azoles. Further studies are warranted to continue to monitor for azole-resistant A. fumigatus within the United States.

INTRODUCTION

Aspergillus species are common causes of diverse fungal infections, ranging from allergic bronchopulmonary aspergillosis to invasive pulmonary disease. Mortality rates can range between 30% and 90% in immunocompromised individuals, including those with profound and prolonged neutropenia, hematopoietic stem cells transplant (HSCT) recipients, solid organ transplant (SOT) recipients, especially lung allografts, and those with hematologic malignancies receiving intensive chemotherapy (1, 2). In addition, breakthrough infections caused by Aspergillus species in patients receiving antifungal prophylaxis or treatment is a major concern (3). Invasive pulmonary aspergillosis can also occur in patients with COVID-19 or severe influenza who require mechanical ventilation (4). Chronic pulmonary aspergillosis is also a major issue in certain groups, including those with previous tuberculosis and sarcoidosis (5, 6), and these patients often require very prolonged therapy. Worldwide, the predominant cause of these infections are members of Aspergillus section Fumigati, which is comprised of over 60 species including Aspergillus fumigatus, the most well-known member of this section (7).

Patient outcomes have improved over the last 2 decades with the availability of voriconazole, as well as with other azoles with activity against Aspergillus species, including posaconazole and isavuconazole, and these triazoles are the mainstays in the treatment of acute invasive and chronic aspergillosis (1, 8). However, the incidence of infections caused by azole-resistant isolates has markedly increased over the last 15 years, and this is now considered an emerging problem worldwide (9, 10). Azole resistance can develop with prolonged clinical azole use or through environmental exposure of the fungi to azole-like compounds (11–13). The predominant mechanisms associated with triazole resistance include point mutations within the CYP51A gene leading to nonsynonymous amino acid changes in the Cyp51 enzyme, which is responsible for the last step in ergosterol biosynthesis and is targeted by this class of antifungals. In addition, tandem repeats of base pairs in the promoter region of CYP51A in association with such mutations have been found with environmental exposure to azole-like compounds (14, 15). Although azole-resistant A. fumigatus is increasing globally, there are marked differences in rates between different geographical locations, with the highest rates of clinical infections caused by resistant strains being reported in parts of Europe (10). Although azole-resistant A. fumigatus has been reported in both clinical and environmental isolates in the United States, the rate of azole-resistance in clinical isolates has not been well-documented. Our objective was to evaluate the species distribution and antifungal susceptibility profiles of a large collection of Aspergillus section Fumigati clinical isolates from institutions across the United States over a multiyear period. We also sequenced the CYP51A gene of phenotypically resistant isolates for mutations associated with azole resistance.

MATERIALS AND METHODS

Fungal isolates.

Aspergillus isolates received for fungal species identification or antifungal susceptibility testing by the Fungus Testing Laboratory at the University of Texas Health Science Center at San Antonio between October 2015 and January 2020 were included. Isolates that had been cultured from animals or environmental sampling were excluded, as were those cultured from nails, ears, and those for which the site of culture was not specified. All isolates were subcultured onto potato flake agar (PFA) the day of receipt prior to further testing.

Species identification.

Species identification was performed by combined phenotypic characteristics (i.e., colony and microscopic morphology and temperature studies) and DNA sequence analysis. Temperature studies were performed by incubating PFA cultures for 3 days at temperatures of 10°C, 45°C, and 50°C and observed for the presence or absence of growth (16). For DNA sequence analysis, individual isolates were suspended in 2-mL vials containing 0.5 mL of 0.1- and 0.5-mm beads (Zymo Research Corporation, Irvine, CA) and 0.7 mL of Buffer G2 (Qiagen, Valencia, CA). Isolates were then lysed using a bead beater instrument (18 cycles of 60 s at 5,000 rpm with a 30-s rest in between each) (Precellys Evolution; Bertin Instruments, Rockville, MD) followed by the addition of proteinase K and incubation at 56°C (17, 18). DNA was then extracted using an EZ1 DNA tissue kit with a BioRobot EZ1 instrument (Qiagen). Sequencing of the partial β-tubulin (BenA) and calmodulin (CaM) genes was performed using the primer pairs Bt2a (5-TGACCCAGCAGATGTT-3′) and Bt2b (5′-GTTGTTGGGAATCCACTC-3′) and CF1L (5-GCCGACTCTTTGACYGARGAR-3) and CF4 (5-TTTYTGCATCATRAGYTGGAC-3), respectively (19). BLASTn searches of these sequences were then performed in GenBank, and results were considered significant with an E-value of 0.0 at 98 to 100% identity and with at least 90% query coverage.

In vitro susceptibility.

Antifungal susceptibility testing was performed for eight different antifungals, including amphotericin B, voriconazole, posaconazole, isavuconazole, itraconazole, anidulafungin, caspofungin, and micafungin, by broth microdilution according to the Clinical and Laboratory Standards Institute (CLSI) M38 standard (20). Briefly, stock solutions of each drug were prepared by dissolving the powders in dimethyl sulfoxide (DMSO), followed by further dilutions in RPMI buffered with 0.165 M morpholinepropanesulfonic acid (MOPS) (pH 7.0), with 0.2% glucose and phenol red, and without bicarbonate. The final concentrations ranges were 0.03 to 16 μg/mL for amphotericin B, voriconazole, posaconazole, isavuconazole, and itraconazole, and 0.015 to 8 μg/mL for each of the echinocandins. The final DMSO concentration in the assay was 1% vol/vol. MICs were read visually at 100% growth inhibition after 48 h of incubation at 35°C for amphotericin B and the triazoles. For the echinocandins, the minimum effective concentration (MEC) was read as the lowest concentration that resulted in morphologic changes (e.g., short, stubby, abnormally branched hyphae) after 24 h of incubation (20, 21). Hamigera insecticola ATCC MYA-3630 (previously identified as Paecilomyces variotii) served as the quality control isolate and was included in each day of testing.

CYP51A gene sequence analysis.

The entire CYP51A gene and its promoter region were also sequenced in isolates that were identified as being phenotypically resistant to voriconazole per the CLSI clinical breakpoint or non-wild type to posaconazole or isavuconazole by published epidemiological cutoff values (ECVs) (21–23). DNA was extracted as described above, and previously published primers and methods were used for sequencing (Table 1) (24–27). Sequences were compared to the CYP51A reference sequence from A. fumigatus strain ATCC 36607 (GenBank accession no. AF222068).

TABLE 1.

Primers used to sequence CYP51A gene and promoter region

Primer	Sequence	Sense or antisense	Region of interest amplified (source)
CYP51A-TR-S1	5′-GGA-GAA-GGA-AAG-AAG-CAC-TCT-3′	Sense	Promoter (24, 27)
CYP51A-TR-R	5′-TCT-CTG-CAC-GCA-AAG-AAG-AAC-3′	Antisense
CYP51A-PMF	5′-GTT-CTT-CTT-TGC-GTG-CAG-AG-3′	Sense	Internal (24, 25)
CYP51A-2R	5′-CCT-TGC-GCA-TGA-TAG-AGT-GA-3′	Antisense
CYP51A-3L	5′-TTC-CTC-CGC-TCC-AGT-ACA-AG-3′	Sense	Internal (26)
CYP51A-3R	5′-CCT-TTG-AAG-TCC-TCG-ATG-GT-3′	Antisense

Data analysis.

Descriptive statistics were used to describe species distributions and sites of culture. MIC/MEC ranges, the MIC/MEC values that inhibited 50% and 90% of isolates (MIC/MEC₅₀ and MIC/MEC₉₀, respectively), and geometric mean (GM) MIC/MEC values were calculated. MIC/MEC values greater than the highest concentration tested were assigned a value 1 dilution higher for the purpose of statistical comparisons. The correlations between isavuconazole, posaconazole, and voriconazole MICs were assessed by Pearson correlation using log₂-transformed MIC values.

RESULTS

Species distribution and sites of infection.

During the 52-month period, 2,138 Aspergillus section Fumigati clinical isolates were included. By far, Aspergillus fumigatus sensu stricto (referred to from hereon as A. fumigatus) was the most prevalent species, accounting for over 96% of the isolates (Table 2). Aspergillus lentulus and Aspergillus hiratsukae were the next most common, but combined, these species accounted for just under 2% of isolates (1.36 and 0.61%, respectively). The lower and upper respiratory tracts (41.9% and 39.9%, respectively) were the most predominant sites from which the isolates were cultured (Fig. 1). Other sites from which at least 2% of isolates were cultured included the eyes/orbital areas, extrapulmonary/pleural fluid, the extremities, and the central nervous system (CNS). Other less common sites included abscesses, the abdomen/gastrointestinal tract, cardiovascular tissue, blood and urine, and bone.

TABLE 2.

Species distribution among 2,138 clinical isolates within Aspergillus section Fumigati^a

Species	No. of isolates	Percentage
Aspergillus fumigatus	2,072	96.91
Aspergillus lentulus	29	1.36
Aspergillus hiratsukae	13	0.61
Aspergillus thermomutatus	8	0.37
Aspergillus udagawae	6	0.28
Aspergillus fumigatiaffinis	4	0.19
Aspergillus fumisynnematus	2	0.09
Aspergillus fischeri	1	0.05
Aspergillus nishimurae	1	0.05
Aspergillus pseudoviridinutans	1	0.05
Aspergillus viridinutans	1	0.05

^aAll isolates were identified to the species level by combined DNA sequence analysis and phenotypic characteristics.

FIG 1.

Site of culture of 2,138 Aspergillus section Fumigati clinical isolates between October 2015 and January 2020 from institutions across the United States.

Antifungal susceptibility.

Antifungal susceptibility testing was performed by broth microdilution testing according to the CLSI methods for amphotericin B, the azoles, and the echinocandins. The MIC results for each antifungal against all isolates tested within this section are shown in Table 3. Amphotericin B demonstrated activity against the majority of isolates tested, with an MIC range of 0.06 to 4 μg/mL and a GM MIC of 0.861 μg/mL. Elevated MICs (4 μg/mL) were observed against 23 isolates, of which 14 were against A. fumigatus (14 of 1,592 isolates tested; 0.88%), 5 were against A. lentulus (5 of 21; 23.8%), and 3 against Aspergillus udagawae (3 of 6; 50%). Of the azoles tested, the lowest MICs were observed with posaconazole, followed by itraconazole, voriconazole, and isavuconazole. MIC values of 8 μg/mL or higher were observed for each of the azoles against several A. fumigatus isolates, indicating high-level azole resistance. Using the CLSI clinical breakpoints of ≥2 μg/mL for voriconazole resistance (21), 3.33% of A. fumigatus isolates were classified as resistant during this 52-month study period, while 91.43% were susceptible (MIC, ≤0.5 μg/mL) and 5.24% were intermediate (MIC, 1 μg/mL). Although CLSI has not established a clinical breakpoint for posaconazole, using the epidemiological cutoff value (ECV) of ≤0.5 μg/mL (22), 93.42% of A. fumigatus isolates were classified as wild type (MIC, ≤0.5 μg/mL) and 6.58% as non-wild type. For isavuconazole and itraconazole (ECV, 1 μg/mL for both) (23), the percentages of wild-type isolates were 95.16% and 95.50%, respectively, and for non-wild type, the percentages were 4.85% and 4.50%, respectively. The rate of voriconazole resistance or percentage of A. fumigatus isolates that were non-wild type to posaconazole or isavuconazole remained relatively stable throughout the study period (Fig. 2), with voriconazole resistance ranging from 3.05% to 4.07%. The percentage of isolates that were non-wild type to posaconazole did rise to 7.08% during the second quarter of the 52-month period but then declined to less than half this value for the remainder of the study period. When the voriconazole clinical breakpoint or the ECVs for isavuconazole and posaconazole are applied to all Aspergillus section Fumigati isolates, the percentages of resistant or non-wild-type isolates increased to 4.76%, 6.05%, and 6.86%, respectively.

TABLE 3.

MIC and minimum effective concentration ranges, MIC/MEC₅₀ and MIC/MEC₉₀ values, modal MIC/MECs, and geometric mean MIC/MEC values for amphotericin B, isavuconazole, itraconazole, posaconazole, voriconazole, anidulafungin, caspofungin, and micafungin against Aspergillus section Fumigati clinical isolates^a

Species	Antifungal (no. isolates tested)	MIC/MEC range	MIC/MEC50b	MIC/MEC90b	Modal MIC/MECb	GM MIC/MECb
Aspergillus section Fumigati	Amphotericin B (1,643)	0.06 to 4	1	2	1	0.861
	Isavuconazole (2,131)	≤0.03 to >16	0.5	1	0.5	1.29
	Itraconazole (1,121)	≤0.03 to >16	0.5	1	1	0.385
	Posaconazole (1,195)	≤0.03 to >16	0.125	0.5	0.25	0.123
	Voriconazole (1,952)	≤0.03 to >16	0.5	1	0.5	0.406
	Anidulafungin (99)	≤0.015 to >8	≤0.015	0.06	≤0.015	0.021
	Caspofungin (406)	≤0.015 to >8	0125	0.25	0.125	0.087
	Micafungin (578)	≤0.015 to 0.5	≤0.015	≤0.015	≤0.015	0.016
Aspergillus fumigatus sensu stricto	Amphotericin B (1,592)	0.06 to 4	1	2	1	0.850
	Isavuconazole (2,065)	≤0.03 to >16	0.5	1	0.5	1.29
	Itraconazole (1,090)	≤0.03 to >16	0.5	1	1	0.385
	Posaconazole (1,155)	≤0.03 to >16	0.125	0.5	0.25	0.121
	Voriconazole (1,891)	≤0.03 to >16	0.5	0.5	0.5	0.392
	Anidulafungin (94)	≤0.015 to >8	≤0.015	0.06	≤0.015	0.021
	Caspofungin (395)	≤0.015 to >8	0.125	0.125	0.125	0.086
	Micafungin (554)	≤0.015 to 0.5	≤0.015	≤0.015	≤0.015	0.016
Aspergillus lentulus	Amphotericin B (21)	0.5 to 4	2	4	2	1.59
	Isavuconazole (29)	0.25 to 8	2	4	2	1.86
	Itraconazole (11)	0.125 to >16	0.5	2	0.5	0.730
	Posaconazole (18)	0.06 to 1	0.125	1	0.125	0.182
	Voriconazole (25)	0.25 to >16	2	4	2	2.36
	Anidulafungin (1)	≤0.015	NC	NC	NC	NC
	Caspofungin (1)	2	NC	NC	NC	NC
	Micafungin (8)	≤0.015 to 0.03	NC	NC	NC	NC
Aspergillus hiratsukae	Amphotericin B (12)	0.125 to 4	0.5	2	0.5	0.794
	Isavuconazole (13)	0.06 to 0.5	0.25	0.5	0.25	0.224
	Itraconazole (9)	≤0.03 to 1	NC	NC	NC	NC
	Posaconazole (7)	≤0.03 to 0.25	NC	NC	NC	NC
	Voriconazole (13)	0.06 to 1	0.5	0.5	0.5	0.325
	Caspofungin (1)	0.06	NC	NC	NC	NC
	Micafungin (6)	≤0.015 to 0.03	NC	NC	NC	NC
Other Aspergillus species within section Fumigati	Amphotericin B (18)	0.5 to 4	1	4	1	1.41
	Isavuconazole (24)	0.25 to 8	1	4	4	1.19
	Itraconazole (11)	0.06 to 4	0.5	2	0.5	0.531
	Posaconazole (15)	≤0.03 to 1	0.5	1	0.5	0.272
	Voriconazole (21)	0.25 to 8	1	4	1	1.49
	Anidulafungin (4)	≤0.015 to 0.06	NC	NC	NC	NC
	Caspofungin (8)	≤0.015 to 0.5	NC	NC	NC	NC
	Micafungin (10)	≤0.015	≤0.015	≤0.015	≤0.015	≤0.015

^aMICs for amphotericin B and the azoles were measured after 48 h of incubation at 35°C as the lowest concentration that resulted in 100% inhibition of growth. MECs for the echinocandins were measured after 24 h of incubation at 35°C as the lowest concentration that resulted in morphologic changes (i.e., short, stubby, abnormally branched hyphae).

^bNC, not calculated, as number of isolates tested was less than 10.

FIG 2.

Percent of isolates that were resistant to voriconazole based on the CLSI clinical breakpoint or non-wild type to isavuconazole or posaconazole based on published epidemiologic cutoff values. The 52-month study period was divided into equal 13-month quarters, beginning in October 2015 and ending in January 2020.

Although the number of isolates tested for species other than A. fumigatus was relatively limited, against non-A. fumigatus strains, elevated azole MICs were more frequently observed. Against A. lentulus, and using the clinical breakpoint for voriconazole resistance (≥2 μg/mL) and the ECVs for non-wild type for isavuconazole (≥2 μg/mL) and posaconazole (≥1 μg/mL), 21 of 25 strains (84%) tested had elevated voriconazole MICs, 21 of 29 (72.4%) had elevated isavuconazole MICs, and 3 of 18 (16.7%) had elevated posaconazole MICs. Similar results were observed against Aspergillus thermomutatus (4 of 6 with elevated voriconazole MICs, 5 of 8 for isavuconazole, and 1 of 4 for posaconazole).

Head-to-head comparisons were also made for isavuconazole, posaconazole, and voriconazole against isolates for which all 3 azoles were tested against at least 10 strains (see Table S1 in the supplemental material). The lowest MIC values were observed for posaconazole against all members of Aspergillus section Fumigati, as well for A. fumigatus and A. lentulus, as evident by lower MIC₅₀/MIC₉₀ values and GM MICs. Interestingly, the in vitro activity of posaconazole in the head-to-head comparison was not as affected against A. lentulus as it was for isavuconazole and voriconazole compared to other members of this section as well as A. fumigatus. In addition, the correlation between voriconazole and isavuconazole MIC values (Pearson r value, 0.764) was higher than that between voriconazole and posaconazole (0.517) and also between isavuconazole and posaconazole (0.601). Many isolates that were resistant or non-wild type to voriconazole or isavuconazole were still classified as wild type to posaconazole (45.9% of isolates that were resistant to voriconazole and 54.9% of those were non-wild type to isavuconazole) (Fig. 3). In contrast, the vast majority of isolates that were resistant to voriconazole were also classified as non-wild type to isavuconazole (96.8%).

FIG 3.

MIC comparisons between voriconazole and isavuconazole (A), voriconazole and posaconazole (B), and posaconazole and isavuconazole (C) against Aspergillus fumigatus sensu stricto clinical isolates. MICs for the azoles were measured after 48 hours of incubation at 35°C as the lowest concentration that resulted in 100% inhibition of growth.

Each of the echinocandins also demonstrated good in vitro activity against Aspergillus section Fumigati isolates. This was evident by low GM MEC values for each member of this antifungal class (range, 0.016 to 0.87 μg/mL), which were primarily measured against A. fumigatus. Micafungin demonstrated the lowest GM MEC value (0.016 μg/mL) followed by anidulafungin (0.021 μg/mL) and caspofungin (0.087 μg/mL).

CYP51A mutations.

The CYP51A gene and its promoter were sequenced in 73 A. fumigatus isolates that were phenotypically resistant to isavuconazole, voriconazole, and/or posaconazole, and the resulting amino acid changes are shown in Table 4. Mutations associated with environmental exposure to azoles (i.e., TR₃₄/L98H and TR₄₆/Y121F/T289A) were found in 14 isolates (19.2%). Numerous isolates (41, or 56.2%) contained mutations within CYP51A but without tandem repeats in the promoter region, and several contained multiple previously undescribed variants leading to amino acid changes at multiple codons (Table 4). However, in 18 phenotypically resistant isolates (24.6%), no mutations were detected within the CYP51A gene, including several isolates with isavuconazole and voriconazole MICs as high at 16 μg/mL. Modal MICs for phenotypically resistant isolates without CYP51A mutations were 4 μg/mL for isavuconazole, 2 μg/mL for voriconazole, and 0.25 μg/mL for posaconazole.

TABLE 4.

Cyp51A amino acid changes resulting from CYP51A mutations detected in 73 strains of A. fumigatus that were phenotypically resistant to isavuconazole, voriconazole, and/or posaconazole

Cyp51A amino acid change(s)	No. of strains	Isavuconazole MIC range	Voriconazole MIC Range	Posaconazole MIC range
TR34/L98H	7	4 to >16	2 to 16	0.5 to 2
TR46/Y121F/T289A	6	>16	>16	1 to 2
TR46/Y121F/T289A/N512I	1	>16	>16	0.25
A9T	2	>16	>16	4 to >16
A9T, G434S	1	16	8	4
F46Y, G54R, M172V, E427K	1	1	0.5	4
F46Y, M172V, E427K	3	2 to 4	1 to 4	1 to 2
F46Y, M172V, N248T, D255E, E427K	2	4 to 8	2 to 8	1 to 2
G54E	3	0.5 to 2	0.5 to 2	2 to >16
Y121F	2	2 to 4	1 to 4	0.25 to 1
G138S	3	8 to 16	4 to 16	0.5 to 2
P216L	2	0.5	0.25 to 0.5	1
M220I	2	2	2 to 4	2
M220K	4	2 to 8	2 to 8	2 to >16
M220R	1	0.5	0.25	1
N248K	1	4	2	0.5
M263I	1	8	8	1
I424V	3	2 to 4	2	0.5
G448S	11	1 to >16	2 to >16	0.06 to 1
No Cyp51A amino acid changes	18	0.5 to 16	0.5 to 16	0.125 to 1

DISCUSSION

Aspergillus species are capable of causing a variety of diseases, including invasive aspergillosis, chronic pulmonary aspergillosis, and allergic bronchopulmonary aspergillosis (ABPA). These saprobic fungi are ubiquitous within the environment and primarily enter the host through inhalation into the airways, including the lungs, where disease often occurs. Host immunosuppression, including prolonged and profound neutropenia, corticosteroid use, and the use of immunosuppressive therapies, is a major risk factor for invasive aspergillosis, while preexisting lung disease, such as chronic obstructive pulmonary disease (COPD), tuberculosis, sarcoidosis, and cystic fibrosis, is common in patients with chronic pulmonary aspergillosis or ABPA. Invasive aspergillosis is also known to cause coinfections in patients with severe viral infections, including influenza and COVID-19, with most infections occurring in patients who require mechanical ventilation and corticosteroid treatment. Of the species that are cultured from patients with aspergillosis, A. fumigatus is typically the most common. Other common species include Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, and Aspergillus terreus. However, some studies have reported an increased prevalence of cryptic Aspergillus species (28–33). These findings may be clinically relevant, as some cryptic species have been reported to have reduced susceptibility to currently available antifungals (28, 30–32, 34).

The triazoles voriconazole, posaconazole, and isavuconazole are the mainstays in the treatment and prophylaxis of invasive and chronic aspergillosis, and each can be administered either intravenously or orally. Oral therapy is an important factor in patients who need prolonged therapy, which is often required in the treatment of aspergillosis. These antifungals inhibit the conversion of lanosterol to ergosterol within the fungal cell membrane by targeting the enzyme lanosterol-14α-demethylase, which is referred to as Cyp51 in Aspergillus. However, azole resistance in Aspergillus can negatively impact patient outcomes, and therapeutic options become limited. Resistance can develop with chronic clinical exposure to this class of antifungals, which can cause nonsynonymous point mutations within the CYP51A gene, leading to amino acid changes within certain codons (e.g., G54E, G138, G448S, and M220K, among others). In fact, azole-resistant Aspergillus isolates were first cultured from patients who had received prolonged itraconazole therapy due to chronic pulmonary aspergillosis (35, 36). However, within the past 15 years it has become recognized that azole resistance can also develop with environmental exposure to azoles, which may be used as fungicides in agricultural settings to prevent fungal infections in various crops and to prevent wood rot (15). Invasive aspergillosis due to azole-resistant A. fumigatus isolates has also been documented in patients without previous azole exposure (37–39). In addition to point mutations within CYP51A, these isolates also have repeats in the promoter regions of the CYP51A gene that caused increased expression (e.g., TR₃₄/L98H, TR₄₆/Y121F/T289A, TR₃₄³/L98H, TR₄₆³/Y121F/M172I/T289A/G448S) (40–42). These mutations have now been reported in clinical and environmental isolates of A. fumigatus in multiple countries on several continents (9, 10), including here in the United States. (43–46). While the rates of azole resistance in A. fumigatus isolates varies from country to country, in the United States. this has been reported to be fairly low (∼4%) (47–50). However, more recent data are not available.

In this study, we report specifically on our experience with Aspergillus section Fumigati during a 52-month period ending in January 2020. Similar to what others have found, the majority of isolates were cultured from the respiratory tract, and A. fumigatus was the most prevalent species. Cryptic species were identified, including A. lentulus, A. hiratsukae, A. thermomutatus, and A. udagawae, among others, but combined these represented less than 4% of the isolates. Although other studies have an increased prevalence of cryptic species when all Aspergillus sections are included, the rates of cryptic species within section Fumigati are similar to what we report here. As part of the 6-year, prospective TRANS-NET surveillance study in solid organ transplant recipients and those undergoing hematopoietic stem cell transplantation in the United States and Canada, Balajee et al. reported that, among members of section Fumigati, 93.9% were Aspergillus fumigatus sensu stricto and 6.1% were other species, including 2.7% A. lentulus and 2.0% A. udagawae (28). Similarly, in a population-based survey of filamentous fungi in Spain, although cryptic species accounted for up to 12% of all Aspergillus species, within section Fumigati, 3.7% were non-Aspergillus fumigatus sensu stricto (29). Similarly, rates of cryptic species within section Fumigati isolates have also been reported by others (31–33). As others have reported, elevated azole and amphotericin B MICs were observed against several non-A. fumigatus isolates in our study. However, as the number of cryptic species was rather limited, further studies are needed to understand the differences in susceptibility profiles that were observed.

Against A. fumigatus, both amphotericin B and the echinocandins demonstrated relatively consistent in vitro activity, with a small number of isolates having elevated MIC values. Overall, resistance to voriconazole in A. fumigatus ranged from 3.05% to 4.07%, with minor fluctuations from year to year. Similar rates were observed for isavuconazole and posaconazole for non-wild-type isolates. During the second 13-month quarter of the study, the percentage of isolates that was non-wild type to posaconazole was 7.08%. However, this may be an outlier, as the rates in subsequent periods were again lower (3.38% and 2.87%). In 73 isolates that were resistant to voriconazole or non-wild type to isavuconazole or posaconazole, we also sequenced the CYP51A gene and its promoter region. Building upon our previous experience with isolates from the United States, several isolates that harbored TR₃₄/L98H or TR₄₆/Y121F/T289A mutations associated with environmental exposure to azoles were identified, as well as numerous isolates with mutations within CYP51A but without tandem repeats in the promoter region of this gene. Several isolates that harbored multiple mutations within CYP51A were also found. High-level voriconazole and isavuconazole resistance was uniformly observed in isolates with the TR₄₆/Y121F/T289A mutation. Overall, approximately a quarter of the isolates that were phenotypically resistant to at least one azole were found to have wild-type CYP51A sequences, which is similar to our previous experience and those of others (37–39, 43). Interestingly, one of the highest posaconazole MICs (>16 μg/mL) was found in an isolate harboring an A9T codon change, which has not previously been associated with high-level azole resistance (51, 52). In fact, the MICs for each of the three azoles was elevated (range, 4 to >16 μg/mL) in the two isolates that only contained the A9T codon change, suggesting that other non-CYP51A-mediated mechanisms may be causing resistance in these isolates. Additional novel CYP51A variants are described within the azole-resistant isolates presented here (Table 4), including M263I (1 isolate), I424V (3 isolates), and G434S (1 isolate). These novel variants have not been further characterized in wild-type isolates, so their specific role in azole resistance versus normal fungal sequence variation needs further study. Mechanisms of resistance other than mutations within CYP51A in A. fumigatus include overexpression of efflux pumps (e.g., AfuMdr4 and Cdr113), gain-of-function mutations in transcription factors (e.g., P88L substitution in the CCAAT-binding transcription factor HapE), mutations in regulatory and sterol biosynthesis elements (e.g., SRBA), and mutations within the 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase-encoding gene (HMG1), which encodes HMG-coenzyme A (CoA) reductase, a rate-limiting enzyme in the ergosterol biosynthetic pathway (53–60). Regardless of the absence or presence of mutations, the MIC values of voriconazole and isavuconazole were in close agreement, and a high percentage of isolates that were resistant to voriconazole were also non-wild type to isavuconazole.

Although our work represents a species distribution and in vitro susceptibility study of Aspergillus section Fumigati within the United States, it is not without limitations. No clinical outcome data are available; thus, we are unable to correlate the MIC results with responses or failures of therapy. While the echinocandins did demonstrate good in vitro activity, it must be remembered that the endpoint for this class of agents against filamentous fungi, such as Aspergillus, is a change in morphology, not an inhibition of growth, and these antifungals are not recommended as first-line therapy for the treatment of aspergillosis (1). We were also unable to determine if there are geographic regions within the United States where azole-resistant or non-wild-type isolates may be more prevalent, since many of our isolates were received from other commercial or reference laboratories. In addition, we did not evaluate for other mechanisms of azole resistance other than CYP51A mutations in isolates that were phenotypically resistant to voriconazole or non-wild type to isavuconazole or posaconazole.

In conclusion, our results suggest that, as a whole, the percentage of A. fumigatus isolates in the United States that are resistant to voriconazole or non-wild type to isavuconazole or posaconazole remains relatively low, and we did not see a trend in reduced susceptibility during this multiyear study. In addition, cryptic species within section Fumigati that have been associated with reduced azole susceptibility were found in small numbers. However, these results may not be predictive of the local epidemiology at institutions that treat patients with risk factors for invasive or chronic aspergillosis. Clinicians and clinical microbiology laboratories need to be aware that azole resistance in A. fumigatus does occur within the United States, and further studies are needed to continue to monitor for increased azole resistance.