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All serious fungal infections need appropriate antifungal therapy for successful patient outcome. Only a few classes of antifungal drugs are available, so the emergence of resistance to single drug classes and now multidrug resistance greatly hampers patient management. Azole resistance among Candida and Aspergillus species is one of the greatest challenges to clinical success, followed by echinocandin and multidrug resistance among some Candida species, especially Candida glabrata. The spread of agriculturally derived azole-resistant Aspergillus fumigatus and emerging threats such as multidrug resistant Candida auris are also alarming. The molecular mechanisms that cause drug resistance are naturally occurring in less susceptible species and are acquired in strains of susceptible organisms. Drug resistance mechanisms include altered drug-target interactions, reduced cellular drug concentrations mediated by drug efflux transporters, and permeability barriers associated with biofilms. Although C auris is inherently multidrug resistant, other strains typically develop resistance through stepwise selection of multiple drug-resistance mechanisms. Cellular stress induced by drug treatment promotes adaptation, which contributes to breakthrough resistance. Drug exposure also drives the emergence of resistance. An effective antifungal stewardship programme is essential to control drug resistance, and should incorporate rapid fungal diagnostics, therapeutic drug monitoring, and clinical intervention teams. The development of better diagnostic tools and strategies that allow targeted use of antifungals is essential to preserve drug effectiveness.

Like severe influenza, coronavirus disease-19 (COVID-19) resulting in acute respiratory distress syndrome (ARDS) has emerged as an important disease that predisposes patients to secondary pulmonary aspergillosis, with 35 cases of COVID-19 associated pulmonary aspergillosis (CAPA) published until June 2020. The release of danger-associated molecular patterns during severe COVID-19 results in both pulmonary epithelial damage and inflammatory disease, which are predisposing risk factors for pulmonary aspergillosis. Moreover, collateral effects of host recognition pathways required for the activation of antiviral immunity may, paradoxically, contribute to a highly permissive inflammatory environment that favors fungal pathogenesis. Diagnosis of CAPA remains challenging, mainly because bronchoalveolar lavage fluid galactomannan testing and culture, which represent the most sensitive diagnostic tests for aspergillosis in the ICU, are hindered by the fact that bronchoscopies are rarely performed in COVID-19 patients due to the risk of disease transmission. Similarly, autopsies are rarely performed, which may result in an underestimation of the prevalence of CAPA. Finally, the treatment of CAPA is complicated by drug–drug interactions associated with broad spectrum azoles, renal tropism and damage caused by SARS-CoV-2, which may challenge the use of liposomal amphotericin B, as well as the emergence of azole-resistance. This clinical reality creates an urgency for new antifungal drugs currently in advanced clinical development with more promising pharmacokinetic and pharmacodynamic profiles.

This article addresses the emergence of echinocandin resistance among Candida species, mechanisms of resistance, factors that promote resistance and confounding issues surrounding standard susceptibility testing. Fungal infections remain a significant cause of global morbidity and mortality, especially among patients with underlying immunosupression. Antifungal therapy is a critical component of patient management for acute and chronic diseases. Yet, therapeutic choices are limited due to only a few drug classes available to treat systemic disease. Moreover, the problem is exacerbated by the emergence of antifungal resistance, which has resulted in difficult to manage multidrug resistant strains. Echinocandin drugs are now the preferred choice to treat a range of candidiasis. These drugs target and inhibit the fungal-specific enzyme glucan synthase, which is responsible for the biosynthesis of a key cell wall polymer. Therapeutic failures involving acquisition of resistance among susceptible organisms like Candida albicans is largely a rare event. However, in recent years, there is an alarming trend of increased resistance among strains of Candida glabrata , which in many cases are also resistant to azole drugs. Echinocandin resistance is always acquired during therapy and the mechanism of resistance is well established to involve amino acid changes in “hot-spot” regions of the Fks subunits carrying the catalytic portion of glucan synthase. These changes significantly decrease the sensitivity of the enzyme to drug resulting in higher MIC values. A range of drug responses, from complete to partial refractory response, is observed depending on the nature of the amino acid substitution, and clinical responses are recapitulated in pharmacodynamic models of infection. The cellular processes promoting the formation of resistant Fks strains involve complex stress response pathways, which yield a variety of adaptive compensatory genetic responses. Stress-adapted cells become drug tolerant and can form stable drug resistant FKS mutations with continued drug exposure. A major concern for resistance detection is that classical broth microdilution techniques show significant variability among clinical microbiology laboratories for certain echinocandin drugs and Candida species. The consequence is that susceptible strains are misclassified according to established clinical breakpoints, and this has led to confusion in the field. Clinical factors that appear to promote echinocandin resistance include the expanding use of antifungal agents for empiric therapy and prophylaxis. Furthermore, host reservoirs such as biofilms in the gastrointestinal tract or intra-abdominal infections can seed development of resistant organisms during therapy. A fundamental understanding of the primary molecular resistance mechanism, along with cellular and clinical factors that promote resistance emergence, is critical to develop better diagnostic tools and therapeutic strategies to overcome and prevent echinocandin resistance.

Prior to the SARS-CoV-2 pandemic, antibiotic resistance was listed as the major global health care priority. Some analyses, including the O’Neill report, have predicted that deaths due to drug-resistant bacterial infections may eclipse the total number of cancer deaths by 2050. Although fungal infections remain in the shadow of public awareness, total attributable annual deaths are similar to, or exceeds, global mortalities due to malaria, tuberculosis or HIV. The impact of fungal infections has been exacerbated by the steady rise of antifungal drug resistant strains and species which reflects the widespread use of antifungals for prophylaxis and therapy, and in the case of azole resistance in Aspergillus , has been linked to the widespread agricultural use of antifungals. This review, based on a workshop hosted by the Medical Research Council and the University of Exeter, illuminates the problem of antifungal resistance and suggests how this growing threat might be mitigated. The impact of fungal infections on human health has been exacerbated by the rise of antifungal drug resistance. In this Review, the authors outline the problem of antifungal resistance and suggest how this growing threat might be mitigated.

Reactive oxidant species (ROS) are unstable, highly reactive molecules that are produced by cells either as byproducts of metabolism or synthesized by specialized enzymes. ROS can be detrimental, e.g., by damaging cellular macromolecules, or beneficial, e.g., by participating in signaling. An increasing body of evidence shows that various fungal species, including both yeasts and molds, increase ROS production upon exposure to the antifungal drugs currently used in the clinic: azoles, polyenes, and echinocandins. However, the implications of these findings are still largely unclear due to gaps in knowledge regarding the chemical nature, molecular origins, and functional consequences of these ROS. Because the detection of ROS in fungal cells has largely relied on fluorescent probes that lack specificity, the chemical nature of the ROS is not known, and it may vary depending on the specific fungus-drug combination. In several instances, the origin of antifungal drug-induced ROS has been identified as the mitochondria, but further experiments are necessary to strengthen this conclusion and to investigate other potential cellular ROS sources, such as the ER, peroxisomes, and ROS-producing enzymes. With respect to the function of the ROS, several studies have shown that they contribute to the drugs’ fungicidal activities and may be part of drug-induced programmed cell death (PCD). However, whether these “pro-death” ROS are a primary consequence of the antifungal mechanism of action or a secondary consequence of drug-induced PCD remains unclear. Finally, several recent studies have raised the possibility that ROS induction can serve an adaptive role, promoting antifungal drug tolerance and the evolution of drug resistance. Filling these gaps in knowledge will reveal a new aspect of fungal biology and may identify new ways to potentiate antifungal drug activity or prevent the evolution of antifungal drug resistance.

Candida glabrata is a major fungal pathogen notable for causing recalcitrant infections, rapid emergence of drug-resistant strains, and its ability to survive and proliferate within macrophages. Resembling bacterial persisters, a subset of genetically drug-susceptible C. glabrata cells can survive lethal exposure to the fungicidal echinocandin drugs. Herein, we show that macrophage internalization induces cidal drug tolerance in C. glabrata , expanding the persister reservoir from which echinocandin-resistant mutants emerge. We show that this drug tolerance is associated with non-proliferation and is triggered by macrophage-induced oxidative stress, and that deletion of genes involved in reactive oxygen species detoxification significantly increases the emergence of echinocandin-resistant mutants. Finally, we show that the fungicidal drug amphotericin B can kill intracellular C. glabrata echinocandin persisters, reducing emergence of resistance. Our study supports the hypothesis that intra-macrophage C. glabrata is a reservoir of recalcitrant/drug-resistant infections, and that drug alternating strategies can be developed to eliminate this reservoir. Arastehfar et al. show that macrophage-engulfed cells of fungal pathogen Candida glabrata exhibit multidrug tolerance and increased survival in the presence of cidal antifungal drugs, forming a reservoir from which drug-resistant mutants emerge

  Factors that contribute to this disease burden and high mortality include insufficient diagnostics and treatment, and the increasing numbers of individuals with immune system defects who are particularly susceptible to fungal pathogens [1]. Because of the high risk of fungal infections in immunocompromised individuals--e.g., low-birth-weight neonates or patients undergoing organ transplantation or chemotherapy--it has become common medical practice to utilize antifungal prophylaxis in these patients during immunosuppression [2-4]. [...]data from the clinic indicating rapid development of drug resistance following initiation of therapy [39] support the notion that exposure of C. glabrata to an azole may create fungal cells with highly variable, \"evolvable\" genomes capable of quick breakthrough of resistance to any other types of drug, such as echinocandins (Fig. 1). [...]patients encountering fluconazole prophylaxis may show infections with azole resistant C. glabrata followed by rapid breakthrough on echinocandin therapy, resulting in multidrug-resistant strains.

Candida glabrata has thoroughly adapted to successfully colonize human mucosal membranes and survive in vivo pressures. prior to and during antifungal treatment. Out of all the medically relevant Candida species, C. glabrata has emerged as a leading cause of azole, echinocandin, and multidrug (MDR: azole + echinocandin) adaptive resistance. Neither mechanism of resistance is intrinsic to C. glabrata, since stable genetic resistance depends on mutation of drug target genes, FKS1 and FKS2 (echinocandin resistance), and a transcription factor, PDR1, which controls expression of major drug transporters, such as CDR1 (azole resistance). However, another hallmark of C. glabrata is the ability to withstand drug pressure both in vitro and in vivo prior to stable “genetic escape”. Additionally, these resistance events can arise within individual patients, which underscores the importance of understanding how this fungus is adapting to its environment and to drug exposure in vivo. Here, we explore the evolution of echinocandin resistance as a multistep model that includes general cell stress, drug adaptation (tolerance), and genetic escape. The extensive genetic diversity reported in C. glabrata is highlighted.

The high clinical mortality and economic burden posed by invasive fungal infections (IFIs), along with significant agricultural crop loss caused by various fungal species, has resulted in the widespread use of antifungal agents. Selective drug pressure, fungal attributes, and host- and drug-related factors have counteracted the efficacy of the limited systemic antifungal drugs and changed the epidemiological landscape of IFIs. Species belonging to Candida, Aspergillus, Cryptococcus, and Pneumocystis are among the fungal pathogens showing notable rates of antifungal resistance. Drug-resistant fungi from the environment are increasingly identified in clinical settings. Furthermore, we have a limited understanding of drug class-specific resistance mechanisms in emerging Candida species. The establishment of antifungal stewardship programs in both clinical and agricultural fields and the inclusion of species identification, antifungal susceptibility testing, and therapeutic drug monitoring practices in the clinic can minimize the emergence of drug-resistant fungi. New antifungal drugs featuring promising therapeutic profiles have great promise to treat drug-resistant fungi in the clinical setting. Mitigating antifungal tolerance, a prelude to the emergence of resistance, also requires the development of effective and fungal-specific adjuvants to be used in combination with systemic antifungals.

The recent global pandemic of COVID-19 has predisposed a relatively high number of patients to acute respiratory distress syndrome (ARDS), which carries a risk of developing super-infections. Candida species are major constituents of the human mycobiome and the main cause of invasive fungal infections, with a high mortality rate. Invasive yeast infections (IYIs) are increasingly recognized as s complication of severe COVID-19. Despite the marked immune dysregulation in COVID-19, no prominent defects have been reported in immune cells that are critically required for immunity to Candida. This suggests that relevant clinical factors, including prolonged ICU stays, central venous catheters, and broad-spectrum antibiotic use, may be key factors causing COVID-19 patients to develop IYIs. Although data on the comparative performance of diagnostic tools are often lacking in COVID-19 patients, a combination of serological and molecular techniques may present a promising option for the identification of IYIs. Clinical awareness and screening are needed, as IYIs are difficult to diagnose, particularly in the setting of severe COVID-19. Echinocandins and azoles are the primary antifungal used to treat IYIs, yet the therapeutic failures exerted by multidrug-resistant Candida spp. such as C. auris and C. glabrata call for the development of new antifungal drugs with novel mechanisms of action.