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Impact of COVID-19 on the antifungal susceptibility profiles of isolates collected in a global surveillance program that monitors invasive fungal infections

Abstract Studies demonstrated the impact of the COVID-19 pandemic in the prevalence and susceptibility profiles of bacterial and fungal organisms. We analyzed 4821 invasive fungal isolates collected during 2018, 2019, and 2020 in 48 hospitals worldwide to evaluate the impact of this event in the occurrence and susceptibility rates of common fungal species. Isolates were tested using the CLSI broth microdilution method. While the percentage of total isolates that were C. glabrata (n = 710 isolates) or C. krusei (n = 112) slightly increased in 2020, the percentage for C. parapsilosis (n = 542), A. fumigatus (n = 416), and C. lusitaniae (n = 84) significantly decreased (P < .05). Fluconazole resistance in C. glabrata decreased from 5.8% in 2018–2019 to 2.0% in 2020, mainly due to fewer hospitals in the US having these isolates (5 vs. 1 hospital). Conversely, higher fluconazole-resistance rates were noted for C. parapsilosis (13.9 vs. 9.8%) and C. tropicalis (3.5 vs. 0.7%; P < .05) during 2020. Voriconazole resistance also increased for these species. Echinocandin resistance was unchanged among Candida spp. Voriconazole susceptibility rates in A. fumigatus were similar in these two periods (91.7% in 2018 and 2019 vs. 93.0% in 2020). Changes were also noticed in the organisms with smaller numbers of collected isolates. We observed variations in the occurrence of organisms submitted to a global surveillance and the susceptibility patterns for some organism-antifungal combinations. As the COVID-19 pandemic is still ongoing, the impact of this event must continue to be monitored to guide treatment of patients affected by bacterial and fungal infections. Lay Summary Secondary infections were documented in COVID-19 patients. We compared the prevalence of invasive fungal isolates consecutively collected in 48 worldwide hospitals and their susceptibility patterns between 2020, the year of the global COVID-19 pandemic, and the two prior years.

To address some of the shortcomings regarding investigations of IFI during the COVID-19 pandemic, we used the SENTRY Antifungal Surveillance Program database to examine isolates of yeasts and molds encountered pre-COVID-19 (2018-2019) and from the COVID-19 (2020) period. The primary objectives of this survey were first, to describe the frequency of isolated fungal species from significant infections in the pre-COVID and COVID periods, and second, to determine the phenotypic and genotypic resistance profiles for the pre-COVID and COVID fungal isolates against systemically active antifungal agents.

Fungal identification
All isolates were submitted to matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) using the MALDI Biotyper (Bruker Daltonics, Billerica, MA, USA) after a purity check. Yeasts that were not identified by these methods were identified using sequencing-based methods for the internal transcribed spacer (ITS) region, 28S ribosomal subunit, or IGS1 for Trichosporon spp. [37][38][39][40] Mold isolates that did not achieve an acceptable identification by MALDI-TOF MS were sequenced for the 28S and 1 of the following genes was analyzed: β-tubulin for Aspergillus spp., translation elongation factor (TEF) for Fusarium spp., or ITS for all other species of filamentous fungi. [37][38][39][40] Susceptibility testing All isolates were tested by broth microdilution methods as described in the M27 41 and M38 42 documents. Interpretive cri-teria, which included clinical breakpoints (CBPs) and epidemiological cut-off values (ECVs) where available, were published in the M59, 43 M60, 44 and M61. 45 Quality control (QC) was performed as recommended by in the M27 41 and M38 42 using C. parapsilosis ATCC 22019, C. krusei ATCC 6258, Aspergillus flavus ATCC 204304, and Aspergillus fumigatus ATCC MYA-3626.

Resistance mechanisms
Candida spp. isolates that exhibited echinocandin MIC values above the ECV and A. fumigatus isolates that displayed azole MIC values above the ECV were subjected to whole genome sequencing. 46 These isolates were selected to ensure that mutations were detected even if present in the tail-end of the wild-type population. DNA regions encoding the FKS hot spots in Candida spp. and CYP regions in A. fumigatus were compared to available sequences in the literature. 46 , 47

Statistical analysis
Differences in percentages were further investigated using the Chi-square test to determine statistical significance ( P < .05).

Frequency of occurrence of fungal pathogens in each age group
Of the 4821 total non-duplicate fungal isolates collected from medical centers worldwide, 3272 were from the pre-COVID survey years 2018-2019 and 1549 were from the COVID survey year 2020 (Table 1 ). The isolates were collected from 48 medical centers located in Europe (2305 isolates from 21 medical centers), North America (1237 isolates from 13 medical centers), the Asia-Pacific region (824 isolates from 9 medical centers), and Latin America (455 isolates from 5 medical centers). All participating centers contributed isolates in each of the three survey years. These isolates were recovered from patients with bloodstream infection (BSI; 2460 isolates), pneumonia in hospitalized patients (PIHP; 757 isolates), skin and skin structure infection (SSSI; 316 isolates), urinary tract infection (UTI; 117 isolates), intraabdominal infection (IAI; 113 isolates), and other infection sites (1058 isolates).
All C. krusei isolates were susceptible to voriconazole ( Table 2 ). MIC 90 values for isavuconazole were ≤1 mg/l for all species in both groups, except for C. guilliermondii isolates from the pre-COVID set (MIC 90 , 4 mg/l). All isolates from the pre-COVID and COVID sets, save one isolate of C. dubliniensis (MIC, 1 mg/l; NWT), exhibited a WT phenotype to amphotericin B. The less common species of Candida identified in both groups included C. fabianii , C. fermentati, C. inconspicua , C. metapsilosis , C. pelliculosa , and C. utilis . Comparable activity was observed for both the echinocandins and triazoles in the COVID and pre-COVID sets (Supplementary Table 1).
Among the isolates of Cryptococcus neoformans var. grubii , the MIC 90 values for each of the nine antifungal agents tested were identical or within a single dilution step for the COVID and pre-COVID sets ( Table 2 ). The ECV for amphotericin B and Cryptococcus neoformans var. grubii was 0.5 mg/l and bisected the MIC distribution in both COVID and pre-COVID sets, resulting in 66.7% NWT for the COVID set and 47.1% NWT for the pre-COVID set ( Table 2 ).
The non-Candida yeasts included five isolates of Cryptococcus gattii SC, four of which were from patients in the pre-COVID period and were WT to fluconazole and voriconazole (Supplementary Tables 3 and 4              Aspergillus fumigatus was the most common species of filamentous fungi isolated from both groups. The vast majority of A. fumigatus ( > 91.0%) were WT to the echinocandins, triazoles, and amphotericin B (Table 2 ). There were 31 isolates that were NWT to one or more of the triazoles, 10 in the COVID set and 21 in the pre-COVID set ( P value = 0.708; Table 2 ). Most of the isolates were from Europe (17/31; 54.8%), followed by North America (9/31; 29.0%) and the Asia-Pacific region (5/31; 16.1%). Most of the NWT isolates of A. fumigatus in the pre-COVID set were from Europe (13/21; 61.9%) compared to only 40% (4/10) in the COVID set. Most of the NWT isolates (20/31; 64.5%) were NWT to more than one triazole, including 5/10 (50.0%) in the COVID set and 15/21 (71.4%) in the pre-COVID set.
Aspergillus section Nigri and section Flavi were tied for second in rank order among the Aspergillus species in both the COVID and pre-COVID sets (12.6%, overall; 12.9%, pre-COVID; 11.7%, COVID). All isolates (100.0%) of Aspergillus section Nigri from both groups were WT to caspofungin, posaconazole, and amphotericin B ( Table 2 ). One isolate from Australia in the COVID set (5.0%) was NWT to itraconazole. Aside from itraconazole, isolates of Aspergillus section Nigri from the COVID set were 100.0% WT to the other antifungal agents tested (Table 2 ). There were three isolates in the pre-COVID set that were NWT to itraconazole (5.0%), one each from Europe, North America, and the Asia-Pacific region. The isolate from Europe was also NWT to voriconazole.
The majority (87.5-100.0%) of Aspergillus section Flavii and Aspergillus section Terrei isolates from both COVID and pre-COVID sets exhibited a WT phenotype for the echinocandins, triazoles, and amphotericin B ( Table 2 and Supplementary Tables  5 and 6). Among the section Flavi , single isolates were NWT to posaconazole in the pre-COVID set and to amphotericin B in the COVID set. Aspergillus section Terrei isolates were all WT to the agents tested, except for the isavuconazole NWT isolates (MIC, 2 mg/L) from the COVID year. Aspergillus section Nidulantes isolates were found in both groups and exhibited similarly low MIC values for the triazoles and echinocandins (Table 2 and Supplementary Tables 5 and 6). Among the less common species of Aspergillus , isolates of Aspergillus lentulus and Aspergillus section Usti were only detected in the pre-COVID set and showed comparably elevated MIC values for all the triazoles (Supplementary Table 6).
The other molds in this survey represented a wide range of species, most of which were isolated only from the pre-COVID set ( Table 2 and Supplementary Tables 7 and 8).

Resistance mechanisms
A total of 48 isolates of Candida spp. were NWT to one or more echinocandin (13, COVID; 35, pre-COVID). The COVID set NWT isolates consisted of 9 isolates of C. glabrata , 3 C. albicans , and 1 C. dubliniensis . Among these isolates, seven (1 C. albicans, 1 C. dubliniensis , and 5 C. glabrata ) were found to harbor a mutation in FKS1 or FKS2 ( Table 3 ). The NWT isolates in the pre-COVID set consisted of 16 isolates of C. glabrata , 5 C. tropicalis , 2 C. albicans , and 12 C. parapsilosis . Mutations in FKS were detected in 12 of 16 isolates of C. glabrata and 1 of 2 isolates of C. albicans (Table 3 ). There were no mutations detected in the NWT isolates of C. tropicalis and C. parapsilosis .
Among echinocandin-NWT C. glabrata , the most frequent mutation in the pre-COVID group of isolates was S663P in FKS2 HS1 (6 isolates), followed by a deletion or alterations of F659 in FKS2 HS1 (6 isolates). Notably, these mutations were not observed in the COVID group. Most of those isolates exhibited diverse mutations in FKS1 HS1, while only one isolate from the pre-COVID group had mutations in this region. All C. albicans and C. dubliniensis isolates displayed a S645P mutation in FKS1 HS1 regardless of the year (Table 3 ). Among the FKS mutants, the majority (55.0%) were from North America (53.8%, pre-COVID; 57.1%, COVID).
There were a total of 31 isolates of Aspergillus fumigatus that were NWT to one or more of the triazoles (10, COVID; 21, pre-COVID). All but two (29/31, 93.5%) A. fumigatus isolates were NWT to itraconazole, 17/31 (54.8%) were NWT to isavuconazole, 13/31 (41.9%) were NWT to voriconazole, and 8/31 (25.8%) were NWT to posaconazole. Among the isolates from the COVID set, 3 of 10 (30%) isolates were found to have  alterations in CYP51A (Table 4 ). Each of the 3 A. fumigatus isolates possessed a different set of alterations and a different geographical origin. The isolate from the US harbored multiple alterations in CYP51A , F46Y, M172V, N248T, D255E, and E427K. The isolate from France carried a Q42L alteration in CYP51B . The isolate from New Zealand carried a single alteration G138C in CYP51A. The NWT isolates from the pre-COVID set included 21 isolates of A. fumigatus , 17 of which (81.0%) carried alterations in CYP51 (Table 4 ). In contrast to the isolates from the COVID set, the most frequent alteration was CYP51A TR34/L98H in the pre-COVID set. This alteration was carried by nine isolates, seven from Italy, one from Belgium, and one from Slovenia. Three isolates from North America, two from the USA and one from Canada, carried the alteration I242V in CYP51A . One North American (USA) isolate carried the CYP51A alteration G448S (NWT to isavuconazole, 47 voriconazole, and itraconazole). A single isolate from Australia carried a CYP51B Q42L alteration and was only NWT to voriconazole. Multiple alterations in CYP51A were detected in isolates from the USA, Czech Republic, and Belgium.
Aside from these A. fumigatus isolates, there was 1 isolate of Aspergillus section Nigri from Europe that was NWT to itra-conazole and voriconazole. This isolate was subjected to whole genome sequencing and was found to harbor a K77Q alteration in CYP51A . An isolate of Aspergillus section Flavi from Thailand was found to be NWT to voriconazole and posaconazole and carried alterations in CYP51A (R5H) and CYP51B (K165E).

Conclusion
COVID-19 presents with a spectrum of disease manifestations ranging from asymptomatic or non-specific flu-like symptoms to pneumonia, sepsis, and life-threatening complications such as ARDS and multiple organ failure. In addition, secondary bacterial and fungal infections have proven to be major risk factors for adverse COVID-19 outcomes. 10 , 11 , 14 Concern for these secondary infectious complications of COVID-19 have resulted in the increased use of empiric antimicrobial therapy with concomitant fears of increased AMR due to drug pressure. 13 , 48 , 49 Whereas resistance to antibacterial agents has garnered most of the attention in the literature, 5 , 10 , 11 , 15 , 16 emergence of antifungal-resistant fungi has largely been confined to reports of single cases of azole-resistant A. fumigatus 32 or azole-and echinocandin-resistant species of Candida following extended exposure to antifungal therapy. 25 , 36 , 50 More recently, invasive infections due to the Mucorales have been documented as COVID-19 complications in select areas of the world. 26 , 33 , 34 Several reports raised concern regarding invasive candidiasis as a complication of severe COVID-19 infection. 18 , 22 , 24 , 25 , 29 , 35 , 36 , 50 These reports describe either clusters of infection (e.g., Candida auris ) or variations in species distribution with the emergence of less common species. 18 , 22 , 24 , 25 , 29 , 35 , 36 , 50 When reported, resistance to antifungal agents during the COVID period is generally described as dependent upon species identification rather than assessment of in vitro resistance. 36 Investigations are required to determine if there are broad shifts in the pathogen distribution and emergence of both acquired and intrinsic antifungal resistance associated with the COVID pandemic.
Although the rank order of the most prominent species of Candida ( C. albicans > C. glabrata > C. parapsilosis > C. tropicalis ) is the same for both pre-COVID and COVID time periods, some differences should be noted. The occurrence of C. glabrata, C. tropicalis, C. dubliniensis , and C. krusei increased during COVID compared to the pre-COVID years. Only C. parapsilosis and C. lusitaniae were less common during COVID than before COVID.
Changes to the in vitro susceptibility of these species are perhaps more interesting than subtle shifts in species. C. glabrata is possibly the one species that appeared to increase in both frequency and antifungal resistance in the past two decades. 51 , 52 This species is well-known for resistance to fluconazole and the other azoles. Most recently, C. glabrata has emerged as resistant to both the azoles and the echinocandin class of agents. 50 , 53 As such, it is notable that although the frequency of C. glabrata as a cause of infection slightly increased during COVID (19.2% of Candida , COVID; 18.0%, pre-COVID), resistance to fluconazole decreased from 5.8 to 2.0% over this time (Table 2 ). We previously noticed this trend 52 and found that it may be influenced by patient age, as resistance to the azoles decreased with patient age. However, there was no relationship between azole resistance and age in the present survey (data not shown).
C. parapsilosis is the one species of Candida that is most closely associated with central venous catheter bloodstream infection and breaks in infection prevention protocols. 54 As such, C. parapsilosis might be a prominent pathogen in the chaos of a COVID ICU. Indeed, fluconazole-resistant isolates of C. parapsilosis originated from the ICU more in the COVID set (52.2%) than pre-COVID (37.8%) set. This species was less frequent in the COVID set than in the pre-COVID set; however, increased resistance to fluconazole was prominent in the COVID set isolates (9.8%, pre-COVID; 13.9%, COVID). Notably, this level of resistance to fluconazole surpasses C. glabrata , a species notorious for decreased susceptibility to the azoles in general. 52 Whereas previously clinicians were fearful of using fluconazole empirically due to the threat of fluconazole-resistant C. glabrata , 52 , 55-57 now it is apparent that other species, namely C. parapsilosis and C. tropicalis, may be becoming more resistant to the azole class of agents than C. glabrata . 52 , 58 These observations underscore the importance identifying Candida to the species level and determining the antifungal susceptibility profile to optimize the care of patients with invasive candidiasis.
C. tropicalis has long been considered one of the most virulent species of Candida , causing severe infections in neutropenic cancer patients. 59 C. tropicalis was the fourth most frequent species of Candida in both pre-COVID and COVID sets. This species, while virulent, has not been especially resistant to any of the antifungal agents. 52 As with C. parapsilosis , resistance to the azole class of agents was more prominent in the COVID set (3.5% resistant to fluconazole) than in the pre-COVID set (0.7% resistant to fluconazole) and exceeded that of C. glabrata (2.0% resistant to fluconazole; Table 2 ).
The finding of emerging resistance to fluconazole among common species of Candida and very low rates of resistance to the echinocandins among species other than C. glabrata provides support for current recommendations regarding the use of echinocandins as an initial empirical therapy for invasive candidiasis pending the results of species identification and antifungal susceptibility testing. 56 Perhaps one of the greatest concerns to treating IFI has been the emergence of resistance to the mold-active triazoles in Aspergillus fumigatus . [60][61][62] Triazole-resistant A. fumigatus has been detected worldwide, but it is most prevalent in Europe. 60 Characterization of the mechanisms of resistance to the azoles in A. fumigatus revealed important patterns relative to the epidemiology of infections. 60 , 62-64 Prolonged drug pressure, such as that seen in the management of chronic bronchopulmonary aspergillosis, 60 , 63 , 64 has been shown to result in several different point mutations in the CYP51 genes. We detected more CYP51 mutant strains of A. fumigatus in the pre-COVID set, likely due to the longer pre-COVID time period despite the comparable frequency of NWT strains in both groups ( Table 2 ).
Although considerably less common than the Candida or Aspergillus species, the non-Candida yeasts and the non-Aspergillus molds present a diverse number of species and a diverse set of antifungal resistance profiles marked by either an intrinsic resistance to the azoles, the echinocandins, or both. [65][66][67] A greater diversity of species of both yeasts and molds were present in the pre-COVID than the COVID set, likely due to the longer pre-COVID survey time. The non-Candida yeasts that were common to both groups include several species with intrinsic resistance to one or more antifungal agent: T. asahi , which is resistant to echinocandins; S. clavata , which is resistant to fluconazole and the echinocandins; and R. mucilaginosa , which is resistant to the azoles and the echinocandins.
Among the non-Aspergillus molds, the Mucorales, Fusarium , Scedosporium , L. prolificans , and Scopulariopsis brevicaulis/S. brumptii represent fungi with intrinsic resistance to one or more antifungal agents. [65][66][67] These pathogens were detected in both pre-COVID and COVID sets, but the individual species were more frequent in the pre-COVID set. Two additional species with resistance to the triazoles were only detected in the pre-COVID set: Microascus cirrosus (MIC, > 8 mg/l for all 4 triazoles) and Rasamsonia argillacea SC (MIC 50 , > 8 mg/l for isavuconazole and voriconazole).
These diverse species of yeasts and molds are frequently difficult to identify with routine methods and exhibit highly variable resistance profiles requiring the use of state-of-the-art species identification methods, such as MALDI-TOF MS or nucleic acid sequencing, as well as antifungal susceptibility testing to optimise infection management. 38 , 65 During the COVID-19 pandemic, invasive infection with Mucorales species has emerged as an infrequent infectious complication relative to CAPA or CAIC. 26 , 33 , 34 Whereas cases of CAM have been reported in several areas of the world, most cases have been reported from India, where diabetes and glucocorticoid exposure in COVID are primary risk factors. 26 Unfortunately, India was not among the participating countries in the SEN-TRY Program. There were few isolates of Mucorales from either group (0.8% of all isolates; 0.9%, pre-COVID; 0.6%, COVID). Posaconazole was the most active of the mold-active triazoles in both groups. Several isolates of Mucorales, including M. circinelloides/ramosissimus , Rhizopus spp., and Mucor sp., were resistant/NWT to posaconazole, itraconazole, and isavuconazole.
There are some limitations to this work that must be acknowledged. First, patient-level data is not collected in SENTRY. Second, patients were not identified as infected with COVID-19 in 2020; rather, we compared isolates from patients hospitalized in the pre-COVID and COVID periods. Third, we did not link the isolation of fungal species and associated resistance profiles with patient presentation, treatment, or outcome. Finally, the SENTRY program is not a population-based survey. Due to the prevalence-based study design of SENTRY (see ref 52 ), each medical center only sends a limited number of isolates each year. As such, the numbers of isolates may reflect what was seen in one part of the year but not necessarily the rest of the year.
In summary, we did not detect any major shift of antifungal resistance among clinical isolates of yeasts and molds from the pre-COVID and COVID periods. Most Candida and Aspergillus species from both eras were susceptible or WT to the azoles, echinocandins, and amphotericin B. The most notable changes in resistance profiles were seen with Candida and the azoles. When pre-COVID and COVID sets were compared, fluconazole resistance and resistance to other the azoles decreased among C. glabrata isolates while it increased among C. parapsilosis and C. tropicalis isolates. Importantly, azole resistance in both C. parapsilosis and C. tropicalis now exceeds that of C. glabrata , precluding the use of simple species identification to guide the use of fluconazole in treating Candida infections. These findings underscore the necessity for accurate species identification and determination of in vitro susceptibility of fungal isolates to optimize the treatment of IFI during the COVID pandemic.