ABSTRACT
The global incidence of mucormycosis has increased in recent years owing to higher numbers of individuals at risk for these infections. The diagnosis and treatment of this aggressive fungal infection are of clinical concern due to differences in species distribution in different geographic areas and susceptibility profiles between different species that are capable of causing highly aggressive infections. The purpose of this study was to evaluate the epidemiology and susceptibility profiles of Mucorales isolates 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, isavuconazole, itraconazole, and posaconazole. During this time frame, 854 isolates were included, representing 11 different genera and over 26 species, of which Rhizopus (58.6%) was the predominant genus, followed by Mucor (19.6%). The majority of isolates were cultured from the upper and lower respiratory tracts (55%). Amphotericin B demonstrated the most potent in vitro activity, with geometric mean (GM) MICs of ≤0.25 μg/ml against all genera with the exception of Cunninghamella species (GM MIC of 1.30 μg/ml). In head-to-head comparisons, the most active azole was posaconazole, followed by isavuconazole. Differences in azole and amphotericin B susceptibility patterns were observed between the genera with the greatest variability observed with isavuconazole. Awareness of the epidemiology of Mucorales isolates and differences in antifungal susceptibility patterns in the United States may aide clinicians in choosing antifungal treatment regimens. Further studies are warranted to correlate these findings with clinical outcomes.
KEYWORDS: mucormycosis, mucoralean fungi, antifungal susceptibility, amphotericin B, posaconazole, isavuconazole, Rhizopus, Mucor, antifungal, Mucorales, susceptibility
INTRODUCTION
Mucormycosis is a destructive and life-threatening invasive fungal infection that is often associated with poor clinical outcomes despite the application of surgery and antifungal therapy. This aggressive mycosis is associated with significant morbidity and mortality, especially in immunocompromised hosts, such as those with poorly controlled diabetes, neutropenic patients, hematopoietic stem cell transplant (HSCT) recipients, and burn and trauma victims (1). These infections are caused by members of the order Mucorales, which is composed of over 250 species in 55 genera. In humans, the major causes of infection belong to the genera Rhizopus, Mucor, and Lichtheimia, followed by Rhizomucor, Cunninghamella, Apophysomyces, and Saksenaea (2, 3). Different species may be recovered more frequently from certain sites of infection. For example, Lichtheimia and Apophysomyces may be more commonly recovered from cutaneous mucormycosis, whereas Cunninghamella species were more common in patients with pulmonary or disseminated infections and are associated with high mortality rates (1, 4).
The global incidence of mucormycosis has increased in recent years, owing to higher numbers of individuals at risk for these infections. A review of the literature by Jeong et al. reported a high burden of mucormycosis in Europe, Asia, and North and South America, with a lower burden in Africa, Australia, and New Zealand (4). Increasingly, these pathogens are recognized as capable of causing infections in otherwise healthy individuals, and outbreaks have been linked to contaminated health care linens, laundry carts, bandages, wooden tongue depressors, and ostomy bag adhesive (5–7). Recently, mucormycosis has garnered attention in both the lay press and medical literature due to numerous reports of coinfections in patients suffering from coronavirus disease 2019 (COVID-19) (termed COVID-19-associated mucormycosis [CAM]) (8–15). Although the antifungals amphotericin B, posaconazole, and isavuconazole are generally active in vitro against members of the order Mucorales, clinical response rates remain suboptimal, and some isolates may have reduced susceptibility to these agents (1, 16–18). In addition, long-term exposure to azoles, particularly voriconazole, and the echinocandins has been linked with breakthrough mucormycosis (4, 19).
Accurate identification to the species level and in vitro susceptibility testing are important to enhance our epidemiological understanding of mucormycosis and to aid in predicting susceptibility patterns, carrying out outbreak investigations, and providing clinicians useful information regarding the appropriateness of therapy. The most recent mucormycosis guidelines from the European Confederation of Medical Mycology and the Mycoses Study Group Education and Research Consortium (ECMM/MSG ERC) strongly recommend diagnosis in tissue by culture or by application of molecular in situ assays (1). The objective of this study was to evaluate the species distribution and antifungal susceptibility profiles of a large collection of Mucorales isolates in U.S. clinical samples over a 52-month period.
MATERIALS AND METHODS
Fungal isolates.
Clinical Mucorales isolates received by the Fungus Testing Laboratory at the University Texas Health Science Center at San Antonio for clinical diagnostic testing (species identification and antifungal susceptibility) from October 2015 through January 2020 were included. Isolates that were cultured from animals or environmental sampling were excluded. All isolates were initially subcultured onto potato flake agar (PFA) the day of receipt prior to further testing.
Species identification.
Species identification was performed by combined phenotypic characterization and DNA sequence analysis. Phenotypic assays included temperature studies and examination of colony and microscopic morphologic characteristics. Mycelia from the isolates were harvested from PFA for genomic DNA extraction as previously described (20, 21). Briefly, portions of the mycelia were suspended in buffer G2 (Qiagen, Valencia, CA) and were lysed using a bead beater instrument (Precellys Evolution, Bertin Instruments, Rockville, MD) followed by the addition of proteinase K and incubation at 56°C. Genomic DNA was then extracted using an EZ1 DNA tissue kit with a BioRobot EZ1 instrument (Qiagen). The internal transcribed spacer rDNA region (ITS) and the D1/D2 domains of the large subunit (LSU) rDNA genes were amplified and sequenced using the primer pairs BMBC-R and NL4R for ITS and D1/D2, respectively (22–24). The sequences were then used to perform BLASTn searches in GenBank, and BLASTn results were considered significant with an E value of 0.0 at 98 to 100% identity and with at least 90% query coverage.
Antifungal agents and in vitro susceptibility.
Antifungal susceptibility testing for amphotericin B, posaconazole, itraconazole (each from Sigma-Aldrich, St. Louis, MO, USA), and isavuconazole (Astellas Pharmaceuticals, Northbrook, IL, USA) was performed by broth microdilution according to the Clinical and Laboratory Standards Institute (CLSI) standard M38 (25). Stock solutions of each drug were prepared by dissolving the powders in dimethyl sulfoxide (DMSO), and further dilutions were prepared in RPMI buffered with 0.165 M MOPS (morpholinepropanesulfonic acid; pH 7.0)–0.2% glucose, without bicarbonate and with phenol red, with the final concentrations for each antifungal ranging from 0.016 to 16 μg/ml. MICs were read visually at 100% inhibition of growth after 24 h of incubation at 35°C for tested drugs. Hamigera insecticola (previously identified as Paecilomyces variotii) ATCC MYA-3630 served as the quality control isolate and was included in each day of testing.
Data analysis.
Species distributions and culture sites were assessed by descriptive statistics. MIC ranges, MIC50s, MIC90s, and geometric mean (GM) MICs were calculated. MICs greater than the highest concentration tested were assigned a value one dilution higher for the purpose of statistical comparisons. The Kruskal-Wallis test with Dunn’s posttest for multiple comparisons and the Mann-Whitney tests were used to assess for significant differences in GM MICs. A P value of ≤0.05 was considered statistically significant.
RESULTS
Species distribution.
During the 52-month period, 854 human clinical isolates were included, representing 11 different genera and 26 species of mucoralean fungi (Fig. 1). The predominant genus was Rhizopus (500/854; 58.5%), of which 40.8% were Rhizopus arrhizus, followed by Rhizopus microsporus (35.2%) and 21.6% Rhizopus delemar, and then other less common Rhizopus species. Mucor was the second most common genus (167/854; 19.6%), of which Mucor circinelloides was the most prevalent (57.5%), followed by Mucor velutinosus (26.3%), Mucor plumbeus (7.8%), Mucor indicus (4.2%), and less common Mucor species. Other genera, such as Lichtheimia, Rhizomucor, Syncephalastrum, Cunninghamella, Saksenaea, Apophysomyces, Blakeslea, Actinomucor, and Poitrasia, accounted for just over 20% of all isolates. The species distributions for each genus are shown in Table S1 in the supplemental material.
FIG 1.
Species distribution of 854 Mucorales isolates identified to the species level by DNA sequence analysis between October 2015 and January 2020 from institutions across the United States.
Culture site.
The upper (30.6%) and lower (24.5%) respiratory tracts were the primary sites from which the isolates were cultured, followed by the upper and lower extremities (11.8% and 10.9%, respectively), the abdomen/gastrointestinal tract (4.2%), bodily fluids (i.e., blood and urine; 3.5%), the orbits (1.8%), and the central nervous system (CNS) (1.6%) (Fig. 2). Thirty-four percent of isolates were recovered from the combined upper respiratory tract/orbital/CNS regions. Of the upper respiratory tract specimens, the majority were Rhizopus species (77.8%), followed by Lichtheimia (9.2%) and Rhizomucor (5.4%). Interestingly, Mucor species were cultured from only 2.3% of upper respiratory tract specimens. In contrast, members of this genus accounted for 20.1% of those cultured from the lower respiratory tract, second only to Rhizopus (52.6%). Other genera from this site, including Syncephalastrum (8.6%), Rhizomucor (8.1%), and Lichtheimia (6.2%), were cultured much less frequently. Both Mucor and Lichtheimia isolates were recovered more frequently from the lower extremities (34.4% and 15.0%, respectively) than from the upper extremities (20.8% and 6.0%, respectively). Apophysomyces was recovered only from the extremities. Similarly, 8 of 11 Saksenaea isolates were also cultured from the extremities. In total, 30 isolates were cultured from the blood or urine, 24 of which were from blood cultures, the majority of which were Mucor species (13 M. velutinosus, 8 M. circinelloides, and 1 M. janssenii). It is unknown if these cultures from the bloodstream or urine represent true infection or culture contamination.
FIG 2.
Sites of isolation of 854 Mucorales isolates between October 2015 and January 2020 from institutions across the United States.
Antifungal susceptibility.
Overall, the most active antifungal against each genus was amphotericin B. Against all isolates for which this polyene was tested, the amphotericin B GM MICs ranged from 0.123 μg/ml against Rhizomucor to 1.39 μg/ml against Cunninghamella species (Table 1). Of the 3 azoles tested, posaconazole was the most active (GM MIC range, 0.157 to 1 μg/ml), followed by itraconazole (GM MIC range, 0.247 to 2 μg/ml) and isavuconazole (GM MIC range, 1.13 to 16 μg/ml). However, it should be noted that itraconazole was the least tested of all of the antifungals included in this study, as it was the least requested by clinicians and laboratories that submitted isolates to our reference laboratory for susceptibility testing. The activity of isavuconazole was markedly lower against Cunninghamella, Mucor, and Syncephalastrum species, where the GM MICs ranged from 8 to 16 μg/ml and the MIC90s were >16 μg/ml. In contrast, isavuconazole had good activity against Rhizopus, Rhizomucor, and Lichtheimia species (GM MIC, 1.13 to 1.46 μg/ml); however, species-specific differences were observed, as described below. Against other genera for which fewer isolates were available, including Saksenaea and Apophysomyces, each antifungal demonstrated good activity, although the itraconazole MIC against one S. vasiformis isolate was >16 μg/ml. For other genera, the numbers of isolates included are too small to make conclusions. Head-to-head comparisons were also made for amphotericin B, isavuconazole, and posaconazole against isolates for which all 3 antifungals were tested (Table 2). Overall, amphotericin B still maintained the best in vitro activity, followed by posaconazole and isavuconazole. Itraconazole was not included in this analysis because of the low number of isolates tested with all antifungals when this azole was included. Epidemiologic cutoff values (ECVs) have also been reported for amphotericin B, posaconazole, and itraconazole against a limited number of Mucorales species (26). The vast majority of isolates in the current study were wild type (range, 96.9% to 100%) (Table S2).
TABLE 1.
MIC ranges, MIC50s, MIC90s, modal MICs, and GM MICs for amphotericin B, isavuconazole, itraconazole, and posaconazole against Mucorales clinical isolatesa
| Genus | Antifungal (no. of isolates tested) | MIC (μg/ml) |
||||
|---|---|---|---|---|---|---|
| Range | 50% | 90% | Modal | GM | ||
| Actinomucor | Amphotericin B (2) | ≤0.03 to 0.25 | ||||
| Isavuconazole (3) | 0.5 to 1 | |||||
| Posaconazole (2) | ≤0.03 | |||||
| Apophysomyces | Amphotericin B (9) | ≤0.03 to 0.125 | ||||
| Isavuconazole (10) | 0.5 to 4 | 2 | 4 | 2 | 2 | |
| Itraconazole (3) | 0.25 to 1 | |||||
| Posaconazole (4) | 0.06 to 0.25 | |||||
| Blakeslea | Amphotericin B (2) | ≤0.03 to 0.125 | ||||
| Isavuconazole (2) | 2 to 8 | |||||
| Itraconazole (1) | 0.125 | |||||
| Posaconazole (1) | 0.5 | |||||
| Cunninghamella | Amphotericin B (17) | 0.5 to 2 | 2 | 2 | 2 | 1.39 |
| Isavuconazole (17) | 4 to >16 | 16 | >16 | >16 | 16 | |
| Itraconazole (7) | 0.25 to 2 | |||||
| Posaconazole (18) | 0.25 to 4 | 0.5 | 2 | 0.25 | 0.673 | |
| Lichtheimia | Amphotericin B (43) | ≤0.03 to 1 | 0.125 | 0.5 | 0.06 | 0.131 |
| Isavuconazole (55) | 0.125 to 8 | 2 | 4 | 1 | 1.46 | |
| Itraconazole (11) | ≤0.03 to 1 | 0.25 | 1 | 1 | 0.247 | |
| Posaconazole (46) | ≤0.03 to 1 | 0.25 | 0.5 | 0.5 | 0.217 | |
| Mucor | Amphotericin B (128) | ≤0.03 to 8 | 0.125 | 0.5 | 0.06 | 0.128 |
| Isavuconazole (142) | 2 to >16 | 8 | >16 | 8 | 8 | |
| Itraconazole (38) | 0.5 to >16 | 4 | >16 | 2 | 2 | |
| Posaconazole (111) | 0.125 to 8 | 1 | 2 | 1 | 1 | |
| Poitrasia | Amphotericin B (1) | ≤0.03 | ||||
| Isavuconazole (1) | >16 | |||||
| Rhizomucor | Amphotericin B (34) | ≤0.03 to 0.5 | 0.125 | 0.5 | 0.06 | 0.123 |
| Isavuconazole (38) | 0.06 to 4 | 2 | 2 | 2 | 1.13 | |
| Itraconazole (8) | 0.06 to 1 | |||||
| Posaconazole (32) | ≤0.03 to 0.5 | 0.125 | 0.5 | 0.25 | 0.157 | |
| Rhizopus | Amphotericin B (362) | ≤0.03 to 2 | 0.25 | 0.5 | 0.25 | 0.182 |
| Isavuconazole (455) | 0.125 to >16 | 1 | 8 | 1 | 1.31 | |
| Itraconazole (111) | 0.06 to >16 | 1 | 4 | 1 | 0.893 | |
| Posaconazole (350) | ≤0.03 to >16 | 0.25 | 1 | 0.25 | 0.259 | |
| Saksenaea | Amphotericin B (7) | ≤0.03 | ||||
| Isavuconazole (7) | 0.25 to 2 | |||||
| Itraconazole (4) | 0.125 to >16 | |||||
| Posaconazole (7) | 0.06 to 0.125 | |||||
| Syncephalastrum | Amphotericin B (24) | ≤0.03 to 1 | 0.06 | 0.5 | 0.03 | 0.097 |
| Isavuconazole (27) | 0.5 to >16 | >16 | >16 | >16 | 9.33 | |
| Itraconazole (11) | 0.125 to 8 | 0.5 | 8 | 0.25 | 0.685 | |
| Posaconazole (20) | 0.06 to 4 | 0.5 | 2 | 0.5 | 0.554 | |
The results of antifungal susceptibilities against all genera are shown. MICs were measured after 24 h of incubation at 35°C as the lowest concentration that resulted in 100% inhibition of growth.
TABLE 2.
MIC ranges, MIC50s, MIC90s, modal MICs, and GM MICs for amphotericin B, isavuconazole, and posaconazole for which results were available for all antifungals against at least 10 clinical isolatesa
| Species (no. tested) | Antifungal | MIC (μg/ml) |
||||
|---|---|---|---|---|---|---|
| Range | 50% | 90% | Modal | GM | ||
| Cunninghamella spp. (all isolates, 16) | Amphotericin B | 0.5 to 2 | 2 | 2 | 2 | 1.35 |
| Isavuconazole | 4 to >16 | >16 | >16 | >16 | >16 | |
| Posaconazole | 0.25 to 1 | 0.5 | 1 | 0.25 | 0.420 | |
| Cunninghamella bertholletiae (11) | Amphotericin B | 0.5 to 2 | 2 | 2 | 2 | 1.37 |
| Isavuconazole | 4 to >16 | >16 | >16 | >16 | 16 | |
| Posaconazole | 0.25 to 1 | 0.5 | 1 | 0.25 | 0.441 | |
| Lichtheimia spp. (all isolates, 40) | Amphotericin B | ≤0.03 to 1 | 0.125 | 0.5 | 0.06 | 0.134 |
| Isavuconazole | 0.125 to 4 | 1 | 4 | 1 | 1.17 | |
| Posaconazole | ≤0.03 to 1 | 0.25 | 0.5 | 0.5 | 0.220 | |
| Lichtheimia corymbifera (16) | Amphotericin B | 0.06 to 0.5 | 0.5 | 0.5 | 0.5 | 0.249 |
| Isavuconazole | 1 to 4 | 2 | 4 | 2 | 2.29 | |
| Posaconazole | 0.125 to 0.5 | 0.5 | 0.5 | 0.5 | 0.310 | |
| Lichtheimia ramosa (22) | Amphotericin B | ≤0.03 to 1 | 0.06 | 0.25 | 0.06 | 0.095 |
| Isavuconazole | 0.125 to 2 | 1 | 2 | 1 | 0.802 | |
| Posaconazole | 0.06 to 1 | 0.125 | 1 | 0.06 | 0.186 | |
| Mucor spp. (all isolates, 106) | Amphotericin B | ≤0.03 to 8 | 0.125 | 0.5 | 0.06 | 0.124 |
| Isavuconazole | 2 to >16 | 8 | >16 | 8 | 8.27 | |
| Posaconazole | 0.125 to 8 | 1 | 2 | 1 | 1.03 | |
| Mucor circinelloides (67) | Amphotericin B | ≤0.03 to 8 | 0.06 | 0.5 | 0.06 | 0.110 |
| Isavuconazole | 2 to >16 | 8 | 16 | 8 | 8.87 | |
| Posaconazole | 0.125 to 8 | 1 | 2 | 1 | 1.17 | |
| Mucor velutinosus (24) | Amphotericin B | ≤0.03 to 0.5 | 0.125 | 0.5 | 0.03 | 0.103 |
| Isavuconazole | 2 to >16 | 4 | 8 | 4 | 3.77 | |
| Posaconazole | 0.25 to 2 | 1 | 2 | 1 | 0.728 | |
| Rhizomucor pusillus (32) | Amphotericin B | ≤0.03 to 0.5 | 0.125 | 0.5 | 0.06 | 0.131 |
| Isavuconazole | 0.06 to 2 | 2 | 2 | 2 | 1.07 | |
| Posaconazole | ≤0.03 to 0.5 | 0.25 | 0.5 | 0.25 | 0.157 | |
| Rhizopus spp. (all isolates, 304) | Amphotericin B | ≤0.03 to 2 | 0.25 | 0.5 | 0.25 | 0.181 |
| Isavuconazole | 0.125 to >16 | 1 | 8 | 1 | 1.23 | |
| Posaconazole | ≤0.03 to >16 | 0.25 | 1 | 0.25 | 0.257 | |
| Rhizopus arrhizus (114) | Amphotericin B | ≤0.03 to 1 | 0.125 | 0.5 | 0.06 | 0.136 |
| Isavuconazole | 0.125 to 4 | 1 | 2 | 1 | 0.907 | |
| Posaconazole | ≤0.03 to 1 | 0.25 | 0.5 | 0.25 | 0.189 | |
| Rhizopus delemar (67) | Amphotericin B | ≤0.03 to 2 | 0.25 | 0.5 | 0.5 | 0.195 |
| Isavuconazole | 1 to 16 | 4 | 16 | 2 | 4.34 | |
| Posaconazole | 0.125 to >16 | 0.5 | 1 | 0.5 | 0.621 | |
| Rhizopus microsporus (121) | Amphotericin B | ≤0.03 to 1 | 0.25 | 1 | 0.25 | 0.227 |
| Isavuconazole | 0.125 to >16 | 1 | 2 | 1 | 0.823 | |
| Posaconazole | ≤0.03 to 4 | 0.25 | 0.5 | 0.25 | 0.213 | |
| Syncephalastrum spp. (19) | Amphotericin B | ≤0.03 to 1 | 0.06 | 0.5 | 0.03 | 0.110 |
| Isavuconazole | 0.5 to >16 | >16 | >16 | >16 | 9.60 | |
| Posaconazole | 0.06 to 4 | 0.5 | 2 | 0.5 | 0.557 | |
MICs were measured after 24 h of incubation at 35°C as the lowest concentration that resulted in 100% inhibition of growth.
Interestingly, species-specific differences in susceptibility patterns were observed in several genera. For Lichtheimia species, significantly higher GM MICs (2.62 to 3.34 times higher) were observed against Lichtheimia corymbifera than Lichtheimia ramosa for amphotericin B (0.249 μg/ml versus 0.095 μg/ml; P < 0.002) and isavuconazole (3.05 μg/ml versus 0.912 μg/ml; P < 0.001) (Table S3). For posaconazole, this difference was also statistically significant (0.311 μg/ml versus 0.177 μg/ml; P = 0.0217) but represented only a 1.76-fold difference in the GM MIC. Similar results were also observed for both isavuconazole and posaconazole against Rhizopus species. Against R. delemar, the GM MICs of both isavuconazole (3.92 μg/ml) and posaconazole (0.581 μg/ml) were significantly higher than those against R. arrhizus (0.902 μg/ml and 0.196 μg/ml, respectively) and R. microsporus (1.03 μg/ml and 0.217 μg/ml, respectively) (P < 0.0001 for all comparisons). These observations of reduced activity for isavuconazole and posaconazole against R. delemar compared to other Rhizopus species explains the wide MIC ranges observed for both azoles against this genus (0.125 to >16 μg/ml for isavuconazole and ≤0.03 to >16 μg/ml for posaconazole). It is unknown if these differences are clinically significant, but they do represent 3.8- to 4.35-times-higher GM MICs for isavuconazole and 2.68- to 2.96-times-higher values for posaconazole against R. delemar compared to the other two Rhizopus species. For isavuconazole, the in vitro activity was also markedly reduced against M. circinelloides (GM MIC 8.68 μg/ml) compared to M. velutinosus (3.77 μg/ml; P < 0.0001).
DISCUSSION
The incidence of mucormycosis, an aggressive and highly destructive invasive fungal infection, has increased in several countries due to increases in the number of at-risk patients (1, 4). Recently, there have also been numerous case reports of mucormycosis in patients suffering from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection (8–11). Although reports have been published from many countries, many of these cases were identified during the COVID-19 surge that occurred in India in the spring of 2021 (12–15). Underlying factors that have been associated with mucormycosis in patients infected with SARS-CoV-2 have included uncontrolled or poorly controlled type II diabetes mellitus and the use of systemic corticosteroids, which in many patients were started prior to the diagnosis of mucormycosis (12, 13, 15). The mortality rates in patients with CAM have varied widely in the literature, ranging from 14% up to 87.5% (8, 13), while many survivors suffer vision loss due to rhinocerebral disease (12, 13).
In many areas, Rhizopus species are the predominant agents of mucormycosis, followed by Mucor, Lichtheimia, Apophysomyces, and Rhizomucor species (1, 27–31). However, Apophysomyces and Lichtheimia species have been reported to be major etiological agents in Asia and some parts of Europe, respectively (1, 4, 19). Recently, cryptic species (e.g., Rhizopus homothallicus and Mucor irregularis) have been reported from Asia (19, 31, 32), and M. velutinosus and Mucor ellipsoideus have been found in the United States, which are relatively uncommon in other geographical regions (27). In a systemic meta-analysis of mucormycosis case reports that included 20 different mucoralean species, Jeong et al. reported that Rhizopus species were the most prevalent (48%), followed by Mucor (14%) and Lichtheimia (13%) species, with R. arrhizus being the most frequently identified individual species (4). However, most Mucor isolates (75%) were not identified to the species level. The overall results of our study are consistent with these findings, as Rhizopus, Mucor, and Lichtheimia species were the most common. However, Mucor isolates were identified to the species level, with M. circinelloides (57.5%) being the most predominant, followed by M. velutinosus (26.3%), M. plumbeus (7.8%), and M. indicus (4.2%). Other species included M. irregularis, Mucor janssenii, and Mucor racemosus (less than 1% each).
The upper respiratory and lower respiratory tracts were the most common sites from which isolates were cultured, followed by the upper and lower extremities. As noted above, approximately a third of isolates were from the combined areas of the upper respiratory tract, orbits, and CNS. These results are also in line with those reported by others. In a previous study in the United States, pulmonary mucormycosis accounted for 24% cases (2), while in France, lung infections have been reported in up to 44% of cases (33). However, dissemination from the lungs may occur frequently (up to 40% in one report) (2). Not surprisingly due to the high prevalence in this study, Rhizopus species were the most frequently cultured from all sites. In a previous systematic review, Rhizopus species were noted more often in patients with rhino-orbital-cerebral mucormycosis, Cunninghamella species were more commonly cultured from pulmonary or disseminated mucormycosis, and Apophysomyces, Lichtheimia, and Saksenaea species were involved mainly in superficial infections, affecting tissues in the upper and lower extremities (4). These observations are also consistent with the results of our study. Interestingly, Mucor isolates were identified in 22 of 24 blood culture isolates. However, we were not able to determine if these represent true infection or contamination of the cultures that may have occurred at the institution from which they originated.
Treatment options for infections caused by the Mucorales are limited due to their intrinsic resistance to several different antifungals, including voriconazole and the echinocandins (1). Often, higher doses of a lipid formulation of amphotericin B are needed, and isavuconazole and posaconazole may be used as alternatives or when transitioning to oral therapy. Antifungal therapy combined with surgical debridement, when feasible, and correction of the underlying immune deficiencies or other risk factors is highly recommended (1). Of the antifungals tested in this study, amphotericin B overall had the most potent and consistent in vitro activity. This was followed by posaconazole and isavuconazole. The activity profile of itraconazole was similar to that of posaconazole, but there were fewer itraconazole MICs available than for the other triazoles. Thus, head-to-head comparisons were not made between itraconazole and the other antifungals. Overall, the results of this study are similar to those previously reported for amphotericin B and the triazoles against Mucorales (26, 34–37). Several species-specific differences in antifungal activity were noted among several species. These differences in potency appeared to affect isavuconazole the most, with higher GM MICs noted against L. corymbifera, M. circinelloides, and R. delemar than other species within these genera, although differences were also observed with amphotericin B and posaconazole. Other studies that have included smaller numbers of isolates have found similar results (34, 36, 37).
Although this study represents one of the largest epidemiologic and in vitro susceptibility studies of the Mucorales, there are some limitations that must be considered. No clinical outcome data were available; thus, we were not able to correlate the in vitro susceptibility results with responses to or failures of therapy. Currently, no antifungal breakpoints have been established by CLSI or EUCAST against any of the Mucorales. It is uncertain how the MIC results, especially the higher values observed against certain species in this study, translate into clinical outcomes. ECVs for amphotericin B, posaconazole, and itraconazole have been published for a few Mucorales species (26), but none are not currently available for isavuconazole. Although itraconazole demonstrated good in vitro activity, the data were limited, as susceptibility testing for this azole is not as often requested of our laboratory. However, in resource-limited settings where access to other orally available antifungals, such as posaconazole and isavuconazole, or intravenous medications, such as amphotericin B formulations, may not be available, itraconazole may be the only treatment option. Also, since many of our isolates were received from other commercial or reference laboratories, we were unable to determine if there may be geographic areas or climates where certain species may be more prevalent. Finally, these results are reflective of isolates that were able to be cultured from patients and thus may not truly reflect the epidemiology of mucormycosis in the United States.
In conclusion, our results build upon those previously published by others and highlight the species diversity and variability in in vitro antifungal susceptibility observed for members of the order Mucorales. Susceptibility testing of Mucorales isolates may be warranted when fungal identifications are not possible within a reasonable time frame or as a means to determine if microbiological failure may be a reason for poor clinical outcomes. However, this may not be needed in all cases in which cultures are available. Clinicians and clinical microbiology laboratories need to be aware of the species distributions and antifungal susceptibility patterns for the Mucorales, as this information may be useful in helping to guide antifungal therapy against highly aggressive infections caused by these species.
ACKNOWLEDGMENTS
N.P.W. has received grant support from Astellas, bioMérieux, Covance, F2G and Sfunga and has served on a Scientific Advisory Board for Mayne Pharma. All other authors report no conflicts.
Funding was received in part from Astellas Pharma, Inc.
Isavuconazole powder was received from Astellas Pharma, Inc.
Footnotes
Supplemental material is available online only.
Contributor Information
Nathan P. Wiederhold, Email: wiederholdn@uthscsa.edu.
Kimberly E. Hanson, University of Utah
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Supplementary Materials
Tables S1 to S3. Download JCM.01230-21-s0001.pdf, PDF file, 0.09 MB (92.3KB, pdf)