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Summary: Background: Mucormycosis is a deadly invasive fungal infection recently included in the WHO priority pathogen list. Here we sought to describe epidemiological trends of mucormycosis in France, and to evaluate factors associated with mortality. Methods: From 2012 to 2022, we implemented a nationwide prospective surveillance programme for mucormycosis in France, focusing on epidemiology, species, seasonal variations. Factors associated with 3-month mortality were studied by univariable and multivariable logistic regression. Findings: Among 550 cases of mucormycosis, the main underlying conditions were haematological malignancy (HM, 65.1%, 358/550), trauma (8%, 44/550), diabetes (7.5%, 41/550) and solid-organ transplants (6.5%, 36/550). Site of infection was pulmonary in 52.4% (288/550), rhinocerebral in 14.5% (80/550), and cutaneo-articular in 17.1% (94/550). Main species identified were Rhizopus arrhizus (21%, 67/316), Rhizopus microsporus (13.6%, 43/316), Lichtheimia corymbifera and Mucor circinelloides (13.3%, 42/316 each), Rhizomucor pusillus (12%, 38/316), and Lichtheimia ramosa (10.8%, 34/316). We found associations between underlying condition, site of infection, and infecting species, including a previously undescribed triad of trauma, cutaneo-articular localisations, and L. ramosa/M. circinelloides. Diagnostic contribution of Polymerase Chain Reaction (PCR) increased from 16% (4/25) in 2012 to 91% (61/67) in 2022, with more than 50% of diagnoses relying solely on PCR in 2022. We also found seasonal variations with relatively more cases in autumn. Ninety-day mortality was 55.8% (276/495). Independent prognostic factors were age, diagnosis in Intensive Care Unit (ICU), and HM while diagnosis after 2015 (i.e. large implementation of PCR) and surgery were associated with reduced mortality. Interpretation: This study reveals major mucormycosis epidemiological changes in France, with a large predominance of HM patients, and a parallel between PCR multicentre implementation and improved prognosis. We also evidence new associations between species, localisations and risk factors, as well as seasonal variations. Funding: Recurrent financial support from Santé Publique France and Institut Pasteur.

ABSTRACTHeteroresistance 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.

ABSTRACT 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.