Approaches for identifying and measuring heteroresistance in azole-susceptible Candida isolates.

Heteroresistance to antifungal agents poses a significant challenge in the treatment of fungal infections. Currently, the absence of established methods for detecting and measuring heteroresistance impedes progress in understanding this phenomenon in fungal pathogens. In response to this gap, we present a comprehensive set of new and optimized methods designed to detect and quantify azole heteroresistance in Candida albicans. Here, we define two primary assays for measuring heteroresistance: population analysis profiling, based on growth on solid medium, and single-cell assays, based on growth in liquid culture. We observe good correlations between the measurements obtained with liquid and solid assays, validating their utility for studying azole heteroresistance. We also highlight that disk diffusion assays could serve as an additional tool for the rapid detection of heteroresistance. These methods collectively provide a versatile toolkit for researchers seeking to assess heteroresistance in C. albicans. They also serve as a critical step forward in the characterization of antifungal heteroresistance, providing a framework for investigating this phenomenon in diverse fungal species and in the context of other antifungal agents. Ultimately, these advancements will enhance our ability to effectively measure antifungal drug responses and combat fungal infections.IMPORTANCEHeteroresistance involves varying antimicrobial susceptibility within a clonal population. This phenomenon allows the survival of rare resistant subpopulations during drug treatment, significantly complicating the effective management of infections. However, the absence of established detection methods hampers progress in understanding this phenomenon in human fungal pathogens. We propose a comprehensive toolkit to address this gap in the yeast Candida albicans, encompassing population analysis profiling, single-cell assays, and disk diffusion assays. By providing robust and correlated measurements through both solid and liquid assays, this work will provide a framework for broader applications across clinically relevant Candida species. These methods will enhance our ability to understand this phenomenon and the failure of antifungal therapy.

Small molecules restore azole activity against drug-tolerant and drug-resistant Candida isolates.

Each year, fungi cause more than 1.5 billion infections worldwide and have a devastating impact on human health, particularly in immunocompromised individuals or patients in intensive care units. The limited antifungal arsenal and emerging multidrug-resistant species necessitate the development of new therapies. One strategy for combating drug-resistant pathogens is the administration of molecules that restore fungal susceptibility to approved drugs. Accordingly, we carried out a screen to identify small molecules that could restore the susceptibility of pathogenic Candida species to azole antifungals. This screening effort led to the discovery of novel 1,4-benzodiazepines that restore fluconazole susceptibility in resistant isolates of Candida albicans, as evidenced by 100-1,000-fold potentiation of fluconazole activity. This potentiation effect was also observed in azole-tolerant strains of C. albicans and in other pathogenic Candida species. The 1,4-benzodiazepines selectively potentiated different azoles, but not other approved antifungals. A remarkable feature of the potentiation was that the combination of the compounds with fluconazole was fungicidal, whereas fluconazole alone is fungistatic. Interestingly, the potentiators were not toxic to C. albicans in the absence of fluconazole, but inhibited virulence-associated filamentation of the fungus. We found that the combination of the potentiators and fluconazole significantly enhanced host survival in a Galleria mellonella model of systemic fungal infection. Taken together, these observations validate a strategy wherein small molecules can restore the activity of highly used anti-infectives that have lost potency. IMPORTANCE In the last decade, we have been witnessing a higher incidence of fungal infections, due to an expansion of the fungal species capable of causing disease (e.g., Candida auris), as well as increased antifungal drug resistance. Among human fungal pathogens, Candida species are a leading cause of invasive infections and are associated with high mortality rates. Infections by these pathogens are commonly treated with azole antifungals, yet the expansion of drug-resistant isolates has reduced their clinical utility. In this work, we describe the discovery and characterization of small molecules that potentiate fluconazole and restore the susceptibility of azole-resistant and azole-tolerant Candida isolates. Interestingly, the potentiating 1,4-benzodiazepines were not toxic to fungal cells but inhibited their virulence-associated filamentous growth. Furthermore, combinations of the potentiators and fluconazole decreased fungal burdens and enhanced host survival in a Galleria mellonella model of systemic fungal infections. Accordingly, we propose the use of novel antifungal potentiators as a powerful strategy for addressing the growing resistance of fungi to clinically approved drugs.

Emergence of azole resistant-Aspergillus fumigatus infections during STAT3-deficiency.

Introduction. Signal transducer and activator of transcription 3 (STAT3) deficiency is a rare primary immunodeficiency associated with increased susceptibility to bacterial and fungal infections, notably pulmonary aspergillosis.Aim. We describe the emergence of azole-resistant Aspergillus fumigatus infections in STAT3-deficient patients.Methodology. During a retrospective study of 13 pulmonary aspergillosis cases in STAT3-deficient patients conducted in France, we identified patients infected with azole-resistant A. fumigatus isolates.Results. Two out of the 13 STAT3-deficient patients with aspergillosis had azole-resistant A. fumigatus infection, indicating an unexpectedly high prevalence of resistance. The first patient with STAT3 deficiency presented several flares of allergic bronchopulmonary aspergillosis-like episodes. He was chronically infected with two azole-resistant A. fumigatus isolates (TR34/L98). Despite prolonged antifungal treatment, including caspofungin and amphotericin B, the patient was not able to clear the azole-resistant A. fumigatus. The second patient had chronic cavitary pulmonary aspergillosis (CCPA). The A. fumigatus isolate was initially azole susceptible but harboured three F46Y, M172V and E427K point mutations. Despite prolonged antifungal therapies, lesions worsened and the isolate became resistant to all azoles. Surgery and caspofungin treatments were then required to cure CCPA. Resistance was probably acquired from the environment (TR34/L98) in the first case whereas resistance developed under antifungal treatments in the second case. These infections required long-term antifungal treatments and surgery.Conclusions. The emergence of azole-resistant A. fumigatus infections in STAT3-deficiency dramatically impacts both curative and prophylactic antifungal strategies. Physicians following patients with primary immune-deficiencies should be aware of this emerging problem as it complicates management of the patient.

Emerging mould infections: Get prepared to meet unexpected fungi in your patient.

Invasive fungal diseases are increasing issues in modern medicine, where the human immunodeficiency virus (HIV) pandemic and the wider use of immunosuppressive drugs generate an ever-growing number of immunocompromised patients with an increased susceptibility to uncommon fungal pathogens. In the past decade, new species have been reported as being responsible for disseminated and invasive fungal diseases in humans. Among them, the following genera are rare but seem emerging issues: Scopulariopsis, Hormographiella, Emergomyces, Westerdykella, Trametes, Actinomucor, Saksenaea, Apophysomyces, and Rhytidhysteron. Delay in diagnosis, which is often the case in these infections, jeopardizes patients' prognosis and leads to increased mortality. Here we summarize the clinical and biological presentation and the key features to identify these emerging pathogens and we discuss the available antifungal classes to treat them. We focused on Pubmed to recover extensively reported human invasive cases and articles regarding the nine previously cited fungal organisms. Information concerning patient background, macroscopic and microscopic description and pictures of these fungal organisms, histological features in tissues, findings with commonly used antigen tests in practice, and hints on potential efficient antifungal classes were gathered. This review's purpose is to help clinical microbiologists and physicians to suspect, identify, diagnose, and treat newly encountered fungi in hospital settings.