Although the federal COVID-19 Public Health Emergency has ended, the long-term impact on healthcare cannot be determined yet. One area of major public health concern is antimicrobial resistance. Opportunistic infections were common in critically ill COVID-19 patients who often required long-term invasive devices (eg, endotracheal intubation, central lines, urinary catheters, etc) and the use of immunomodulatory agents for the treatment of severe disease (eg, systemic corticosteroids, baricitinib, tocilizumab [Actemra, Genentech], etc).1,2 This scenario led to the widespread use of broad-spectrum antimicrobial agents globally. Overcrowded medical facilities and shortages of personal protective equipment due to supply chain issues led to widespread ineffective infection control practices.2 These factors led to an increase in rates of many antimicrobial resistance threats—both bacterial and fungal.2
Antimicrobial resistance is a major concern in medical mycology where antifungal resistance is being seen in both yeasts (Candida species) and molds (Aspergillus and Trichophyton species).3 This is due to a combination of factors, such as increased use of broad-spectrum antifungals in hospitalized patients, overuse of topical antifungal ointments/creams, and environmental exposure to antifungals due to agricultural practices.1,2 This article focuses on the diagnostic and treatment options available for emerging antimicrobial-resistant fungal pathogens, namely antifungal-resistant Candida species, including Candida auris, azole-resistant Aspergillus species, and antimicrobial-resistant dermatophytes.3
Candida species are a particularly concerning antimicrobial-resistance threat. The CDC lists antifungal-resistant Candida species as a serious threat and antifungal-resistant C. auris as an urgent threat.1
During 2020, there was a significant increase in cases of antifungal-resistant Candida species reported by the CDC; an estimated 28,100 cases resulted in 14,000 deaths, a 12% overall and 26% hospital-onset increase from 2019.1 C. auris cases have risen exponentially from 328 reported cases in 2018 to 2,377 clinical cases and 5,754 screening cases reported to the CDC in 2022.4,5
Antifungal-Resistant Fungi: Diagnostic Dilemma
Clinical mycology is a challenging field, as most fungal isolates from nonsterile body sites are often representative of colonization or contamination rather than true pathogenicity. Yeasts are a part of the human microbiome and considered part of the normal flora, especially in the gastrointestinal and genitourinary tracts. Yeast can also be found colonizing the upper respiratory tract but rarely is associated with pneumonia, except for Cryptococcus species. Molds are not as commonly associated with the human microbial flora; however, they are ubiquitous in nature and can find their way into clinical specimens in a variety of ways. Fungal spores can be inhaled leading to growth in respiratory cultures. They can also land on specimens during collection or on agar plates during media inoculation. Given this characteristic, susceptibility testing is not performed routinely on fungal isolates from nonsterile sources, as they often represent benign colonization or contamination of the specimen. Susceptibility testing on fungi can be challenging and is often only performed at reference labs and large academic centers with expertise in this area. These factors can make detecting antifungal-resistant fungi challenging.
Although most laboratories can identify the majority of clinically encountered yeasts, C. auris can be difficult to identify routinely in many testing facilities. Morphological characteristics and biochemical testing alone cannot be used to identify C. auris. Molecular assays and matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF) with an updated reference library are the only reliable definitive testing of clinical isolates currently available.6 In some circumstances, sequencing can be used for confirmation testing of presumptive isolates.7 Whole-genome sequencing has the added benefit of allowing phylogenetic evaluation of the isolate, which may aid in infection control practices.7
Invasive infections from Candida species are associated with significant morbidity and mortality. In patients with candidemia, rapid and accurate detection of Candida species associated with antifungal resistance is of utmost importance. The Biofire FilmArray BCID2 (Blood Culture Identification 2) panel and the GenMark Dx ePlex BCID-FP panel are FDA-approved molecular microarray panels used widely in clinical labs.6 These panels can rapidly and accurately detect commonly resistant yeasts, such as C. glabrata, C. krusei, and most importantly, C. auris (Table 1). Although no resistance markers are provided by these microarray panels, species-level identification is important, as the known resistance patterns of certain species can be applied to treatment decisions (Table 2). This can allow broadening or narrowing of antifungal therapy based on pathogen identification.
| Table 1. Fungal Targets on Blood Culture Identification Multiplex Panels | |
| Multiplex BCID Panel | Targets |
|---|---|
| GenMark Dx ePlex BCID-FP Panel | Candida abicans |
| C. auris | |
| C. dubliniensis | |
| C. famata | |
| C. glabrata | |
| C. guilliermondii | |
| C. kefyr | |
| C. krusei | |
| C. lusitaniae | |
| C. parapsilosis | |
| C. tropicalis | |
| Cryptococcus gattii | |
| Cryptococcus neoformans | |
| Fusarium | |
| Rhodotorula | |
| GenMark Dx ePlex BCID-GP Panel | Pan Candida |
| GenMark Dx ePlex BCID-GN Panel | Pan Candida |
| Biofire FilmArray BCID2 Panel | C. albicans |
| C. auris | |
| C. glabrata | |
| C. krusei | |
| C. parapsilosis | |
| C. tropicalis | |
| Cryptococcus (C. neoformans/C. gatti) | |
| BCID, blood culture identification; GN, gram-negative; GP, gram-positive. | |
| Table 2. Fungal Intrinsic Resistance | |
| Organism | Intrinsic resistance to: |
|---|---|
| Yeasts | |
| Candida krusei | Fluconazole |
| Cryptococcus species | Echinocandins |
| Rhodotorula species | Echinocandins and fluconazole |
| Trichosporon species | Echinocandins |
| Molds | |
| Aspergillus species | Fluconazole |
| Lomentospora prolificans | Amphotericin B |
| Mucorales | Fluconazole |
| Purpureocillium lilacinum | Amphotericin B |
| Organism | Associated with high rates of acquired resistance to: |
| Yeasts | |
| Candida glabrata | Fluconazole |
| C. lusitaniae | Amphotericin B |
| C. auris | Azoles, echinocandins, amphotericin B, flucytosine |
| C. haemulonii complexa | Fluconazole, amphotericin B |
| Molds | |
| Aspergillus fumigatus | Triazoles |
| A. flavus | Triazoles |
| Trichophyton species | Terbinafine |
a Candida haemulonii complex includes C. haemulonii, C. duobushaemulonii, C. pseudohaemulonii, and C. vulturna. Adapted from Table 8.104-5 (Clinical Microbiology Procedures Handbook, | |
The Bruker MBT Sepsityper Kit allows for processing of blood culture bottles to create a microorganism pellet, which can be analyzed by MALDI-TOF for rapid identification.8,9 The two commercially available MALDI-TOF instruments are the Bruker MALDI Biotyper (MBT) and BioMérieux VITEK MS. Accuracy for this method depends on the quality of protein extraction and robustness of the instrument’s reference library. Of note, these tests can identify Candida species associated with resistance but do not detect resistance genes or provide antimicrobial susceptibility data. Culture and susceptibility testing are still required.
As cases of C. auris have risen in the United States, so has the use of colonization screening in high-risk patients. C. auris differs from other yeast species, as it commonly colonizes the skin and mucocutaneous sites of individuals. Due to this phenomenon, swabs collected from the bilateral axilla and groin are the preferred specimen for screening.10 Although patients are often asymptomatic, colonization with C. auris predisposes them to superficial cutaneous infections and invasive disease.11 There are several options for screening. Molecular testing provides accurate testing results with a quick turnaround time. There are several commercially available reagent kits. However, these are not cleared by the FDA and would need to be created as a laboratory-developed test requiring a comprehensive validation study.6 Chromogenic agars are available, such as CHROMagar Candida Plus (ChroMagar) and HardyCHROM Candida+auris (Hardy Diagnostics), which are selective and differentiate media for the presumptive identification of C. auris and other Candida species (Figure).12,13 The added benefit of chromogenic agar over molecular assays is the ability to identify other potentially antimicrobial-resistant Candida species. The downside is the need for prolonged incubation time (48-72 hours) until a final negative result can be determined, as well as the need for confirmatory testing, such as MALDI-TOF.
Laboratory diagnosis of molds still relies heavily on morphological characteristics, especially observation of fruiting bodies. This is a process that can take several weeks depending on the growth characteristics of the organism. The use of MALDI-TOF for identification of filamentous molds can significantly decrease identification time and often provide more detailed information, such as species-level determination, over morphological characterization alone.14 Diagnosis of invasive mold infections can be challenging, as molds do not often undergo sporulation in blood or infected tissues. Fusarium species is an exception, as it undergoes adventitious sporulation. The GenMark Dx ePlex BCID-FP panel contains a target for Fusarium, which can provide a rapid diagnosis for disseminated Fusarium infections. Outside of Fusarium, blood culture collections are low yield for detection of invasive fungal infections. Detection of fungal-specific antigens in blood and body fluids can be helpful in certain clinical scenarios (Table 3).
| Table 3. Examples of Assays That Detect Fungal Antigens | |||
| Assay | Antigen | Specimen source | Diagnostic aide in: |
|---|---|---|---|
| Fungitell assay (Associates of Cape Cod, Inc, E. Falmouth, MA) | (1?3)-ß-D-Glucan | Serum |
|
| Clarus Aspergillus Galactomannan EIA (IMMY, Inc, Norman, OK) | Aspergillus galactomannan | Serum, bronchoalveolar lavage (BAL) | Invasive aspergillosis |
| CrAg LFA (IMMY, Inc, Norman, OK) | Glucuronoxylomannan (GXM) | Serum, CSF | Cryptococcal meningitis |
| Histoplasma GM EIA (IMMY, Inc, Norman, OK) | Histoplasma galactomannan | Urine | Histoplasmosis |
There has been an increase in the use of meta- genomic studies to identify invasive mold infections in at-risk patients.15,16 Cost, turnaround time, and specificity issues limit the widespread use of these metagenomic studies. Detection of resistance in mold isolates usually is determined by susceptibility testing of invasive isolates or recurrent treatment-resistant infections. The application of targeted polymerase chain reaction from formalin-fixed paraffin-embedded tissue may be useful for rapid identification of invasive mold infections if a tissue sample is obtainable.17 Common resistance mechanisms can also be targeted with this technique.17
Resistant Mechanisms in Fungal Pathogens: Evolving Threat
Related Content
Multidrug-resistant isolates of C. auris have been identified throughout the world.5,18 These strains are difficult to treat, as they can be resistant to all 3 major classes of antifungal therapy commonly used for systemic treatment: azoles, echinocandins, and polyenes (amphotericin B). Some pan-resistant strains are also resistant to the antimetabolites class (flucytosine).19 There are 4 major clades of C. auris, each with varying resistance patterns.19 Azole resistance appears to be associated with mutations in the ERG11 and CDR1 genes, while echinocandin resistance is seen in mutations of the FKS gene.19 Mutations in the ERG11 gene, part of the cytochrome P450 family, cause alterations in the structure of lanosterol 14 alpha-demethylase, resulting in a decreased azole-binding affinity for the enzyme.19 Other mutations associated with azole resistance include hapE, hmg1, and multiple mdr genes.18,19 ERG gene mutations also result in cross-resistance to amphotericin B due to decreased production of ergosterol.18,19 Decreased levels of ergosterol lead to ineffective amphotericin B binding across the cell wall, resulting in a reduced pore (ion-channel) formation.18 Overexpression of efflux pumps is associated with mutations in the CDR gene.18 FSK gene mutations result in structural changes to 1,3-beta-D-glucan synthase, resulting in reduced enzymatic binding toward echinocandins.18 Mutations in the FUR1, FCY1, and ADE17 genes are seen in isolates with resistance to flucytosine.19 These genes are key in the conversion of flucytosine into 5-fluorouracil, the active antimetabolite.18,20 Similar mutations described in C. auris are seen in other antimicrobial-resistant Candida species.
The azole drug class is the front-line treatment option for aspergillosis. Azole resistance has emerged, mainly due to mutations in the cyp51A gene of molds, the corresponding gene to ERG11 in yeast.17,21-23 Non-cyp51A mutations resulting in azole resistance have also been described.24 Aspergillus fumigatus and A. flavus are the 2 species where azole resistance is most common.24 Rare reports of azole resistance have been documented in A. terreus, A. niger, and A. tubingensis.23
Susceptibility testing on dermatophytes rarely is performed; therefore, clinical data are scarce and no Clinical & Laboratory Standards Institute clinical breakpoints are available for interpretation. Potentially terbinafine-resistant strains have been reported in Trichophyton species.25 Elevated minimum inhibitory concentration values compared with established epidemiological cutoff values have been observed in treatment-refractory patients—a concerning finding for resistance.25 Mutations in the SQLE gene have been observed in these isolates. The SQLE gene encodes for squalene epoxidase, the enzyme target for terbinafine’s antifungal activity.25
Novel Antifungal Agents: Brief Introduction
Several novel antifungal agents have been developed and may be useful in the treatment of highly resistant fungi. Ibrexafungerp (Brexafemme, Scynexis) is a triterpenoid with FDA approval for treatment of vulvovaginal candidiasis in adult and post-menarchal pediatric females. It is the first orally available 1,3-beta-D-glucan synthase inhibitor and has activity against most Candida species including C. auris.26,27 Rezafungin (Rezzayo, Melinta Therapeutics) is a novel echinocandin with recent FDA approval to treat candidemia and invasive candidiasis in adult patients with limited treatment alternatives.26,28 Fosmanogepix (Pfizer) is a first-in-class broad-spectrum antifungal agent in clinical trials with the potential to treat highly resistant invasive fungal pathogens.29 It is the first glycosylphosphatidylinositol inhibitor and functions by disrupting Gwt1 enzyme function.26 Olorofim (F2G) is another first-in-class antifungal agent currently in clinical trials. It has gained FDA Orphan Drug status and Breakthrough Therapy designation for invasive mold infections and central nervous system coccidioidomycosis when treatment options are limited.26 It belongs to the novel orotomides class and is an inhibitor of dihydroorotate dehydrogenase (pyrimidine synthesis).26
Conclusion
The rise in antimicrobial-resistant fungal pathogens, especially C. auris, is a concerning global issue. Rapid and accurate identification of these organisms is paramount for successful treatment and infection control practice. The use of molecular assays and MALDI-TOF have supplanted morphological and biochemical testing as the quickest, most accurate process of identifying clinically significant fungi. Several novel antifungal agents may provide effective treatment options in the future for invasive infections from highly resistant yeast and molds.
References
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- Martel CM, Parker JE, Warrilow AG, et al. Complementation of a aaccharomyces cerevisiae ERG11/CYP51 (sterol 14a-demethylase) doxycycline-regulated mutant and screening of the azole sensitivity of Aspergillus fumigatus isoenzymes CYP51A and CYP51B. Antimicrob Agents Chemother. 2010;54(11):4920-4923.
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- Shen JJ, Arendrup MC, Verma S, et al. The emerging terbinafine-resistant trichophyton epidemic: what is the role of antifungal susceptibility testing? Dermatology. 2022;238(1):60-79.
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About the author:
Acknowledgment: I would like to thank Paul P. Cook, MD, of the Brody School of Medicine at East Carolina University, in Greenville, North Carolina, for his assistance in article review and revisions.
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Antimicrobial Resistance in Perspective: Using New Technology to Help Identify Resistant Fungal Pathogens
