Novel antifungals and treatment approaches to tackle resistance and improve outcomes of invasive fungal disease.

SUMMARYFungal infections are on the rise, driven by a growing population at risk and climate change. Currently available antifungals include only five classes, and their utility and efficacy in antifungal treatment are limited by one or more of innate or acquired resistance in some fungi, poor penetration into "sequestered" sites, and agent-specific side effect which require frequent patient reassessment and monitoring. Agents with novel mechanisms, favorable pharmacokinetic (PK) profiles including good oral bioavailability, and fungicidal mechanism(s) are urgently needed. Here, we provide a comprehensive review of novel antifungal agents, with both improved known mechanisms of actions and new antifungal classes, currently in clinical development for treating invasive yeast, mold (filamentous fungi), Pneumocystis jirovecii infections, and dimorphic fungi (endemic mycoses). We further focus on inhaled antifungals and the role of immunotherapy in tackling fungal infections, and the specific PK/pharmacodynamic profiles, tissue distributions as well as drug-drug interactions of novel antifungals. Finally, we review antifungal resistance mechanisms, the role of use of antifungal pesticides in agriculture as drivers of drug resistance, and detail detection methods for antifungal resistance.

Ibrexafungerp is efficacious in a neutropenic murine model of pulmonary mucormycosis as monotherapy and combined with liposomal amphotericin B.

Ibrexafungerp (formerly SCY-078) is the first member of the triterpenoid class that prevents the synthesis of the fungal cell wall polymer ß-(1,3)-D-glucan by inhibiting the enzyme glucan synthase. We evaluated the in vivo efficacy of ibrexafungerp against pulmonary mucormycosis using an established murine model. Neutropenic mice were intratracheally infected with either Rhizopus delemar or Mucor circinelloides. Treatment with placebo (diluent control), ibrexafungerp (30 mg/kg, PO BID), liposomal amphotericin B (LAMB 10 mg/kg IV QD), posaconazole (PSC 30 mg/kg PO QD), or a combination of ibrexafungerp plus LAMB or ibrexafungerp plus PSC began 16 h post-infection and continued for 7 days for ibrexafungerp or PSC and through day 4 for LAMB. Ibrexafungerp was as effective as LAMB or PSC in prolonging median survival (range: 15 days to >21 days) and enhancing overall survival (30%-65%) vs placebo (9 days and 0%; P < 0.001) in mice infected with R. delemar. Furthermore, median survival and overall percent survival resulting from the combination of ibrexafungerp plus LAMB were significantly greater compared to all monotherapies (P ≤ 0.03). Similar survival results were observed in mice infected with M. circinelloides. Monotherapies also reduce the lung and brain fungal burden by ~0.5-1.0log10 conidial equivalents (CE)/g of tissue vs placebo in mice infected with R. delemar (P < 0.05), while a combination of ibrexafungerp plus LAMB lowered the fungal burden by ~0.5-1.5log10 CE/g compared to placebo or any of the monotherapy groups (P < 0.03). These results are promising and warrant continued investigation of ibrexafungerp as a novel treatment option against mucormycosis.

A Recombinant Multivalent Vaccine (rCpa1) Induces Protection for C57BL/6 and HLA Transgenic Mice against Pulmonary Infection with Both Species of Coccidioides.

Coccidioidomycosis is caused by Coccidioides posadasii (Cp) and Coccidioides immitis (Ci), which have a 4-5% difference in their genomic sequences. There is an urgent need to develop a human vaccine against both species. A previously created recombinant antigen (rCpa1) that contains multiple peptides derived from Cp isolate C735 is protective against the autologous isolate. The focus of this study is to evaluate cross-protective efficacy and immune correlates by the rCpa1-based vaccine against both species of Coccidioides. DNA sequence analyses of the homologous genes for the rCpa1 antigen were conducted for 39 and 17 clinical isolates of Cp and Ci, respectively. Protective efficacy and vaccine-induced immunity were evaluated for both C57BL/6 and human HLA-DR4 transgenic mice against five highly virulent isolates of Cp and Ci. There are total of seven amino acid substitutions in the rCpa1 antigen between Cp and Ci. Both C57BL/6 and HLA-DR4 mice that were vaccinated with an rCpa1 vaccine had a significant reduction of fungal burden and increased numbers of IFN-γ- and IL-17-producing CD4+ T cells in the first 2 weeks post challenge. These data suggest that rCpa1 has cross-protection activity against Cp and Ci pulmonary infection through activation of early Th1 and Th17 responses.

Tuning sterol extraction kinetics yields a renal-sparing polyene antifungal.

Decades of previous efforts to develop renal-sparing polyene antifungals were misguided by the classic membrane permeabilization model1. Recently, the clinically vital but also highly renal-toxic small-molecule natural product amphotericin B was instead found to kill fungi primarily by forming extramembraneous sponge-like aggregates that extract ergosterol from lipid bilayers2-6. Here we show that rapid and selective extraction of fungal ergosterol can yield potent and renal-sparing polyene antifungals. Cholesterol extraction was found to drive the toxicity of amphotericin B to human renal cells. Our examination of high-resolution structures of amphotericin B sponges in sterol-free and sterol-bound states guided us to a promising structural derivative that does not bind cholesterol and is thus renal sparing. This derivative was also less potent because it extracts ergosterol more slowly. Selective acceleration of ergosterol extraction with a second structural modification yielded a new polyene, AM-2-19, that is renal sparing in mice and primary human renal cells, potent against hundreds of pathogenic fungal strains, resistance evasive following serial passage in vitro and highly efficacious in animal models of invasive fungal infections. Thus, rational tuning of the dynamics of interactions between small molecules may lead to better treatments for fungal infections that still kill millions of people annually7,8 and potentially other resistance-evasive antimicrobials, including those that have recently been shown to operate through supramolecular structures that target specific lipids9.

Coccidioides species antifungal susceptibility testing: Experience from a large healthcare system in the endemic region.

The clinical utility of Coccidioides species antifungal susceptibility testing (AST) remains unclear. This study describes the clinical course of eight patients with severe or chronic coccidioidomycosis and subsequent Coccidioides AST. We present the clinical manifestations, antifungal treatment regimens, and clinical outcomes for these patients.

The role of antifungal susceptibility in the management of coccidioidomycosis remains unknown. This report presents cases of complex coccidioidomycosis where clinicians elected to conduct antifungal susceptibility testing as part of the treatment approach.

High-Throughput Screening of the Repurposing Hub Library to Identify Drugs with Novel Inhibitory Activity against Candida albicans and Candida auris Biofilms.

Candidiasis is one of the most frequent nosocomial infections affecting an increasing number of at-risk patients. Candida albicans remains the most frequent causative agent of candidiasis, but, in the last decade, C. auris has emerged as a formidable multi-drug-resistant pathogen. Both species are fully capable of forming biofilms, which contribute to resistance, increasing the urgency for new effective antifungal therapies. Repurposing existing drugs could significantly accelerate the development of novel therapies against candidiasis. Here, we have screened the Repurposing Hub library from the Broad Institute, containing over 6000 compounds, in search for inhibitors of C. albicans and C. auris biofilm formation. The primary screen identified 57 initial hits against C. albicans and 33 against C. auris. Confirmatory concentration-dependent assays were used to validate the activity of the initial hits and, at the same time, establish their anti-biofilm potency. Based on these results, ebselen, temsirolimus, and compound BAY 11-7082 emerged as the leading repositionable compounds. Subsequent experiments established their spectrum of antifungal activity against yeasts and filamentous fungi. In addition, their in vivo activity was examined in the murine models of hematogenously disseminated C. albicans and C. auris infections. Although promising, further in vitro and in vivo studies are needed to confirm their potential use for the therapy of candidiasis and possibly other fungal infections.

Update on Outbreak of Fungal Meningitis among U.S. Residents who Received Epidural Anesthesia at Two Clinics in Matamoros, Mexico.

BACKGROUND: Public health officials are responding to an outbreak of fungal meningitis among patients who received procedures under epidural anesthesia at two clinics (River Side Surgical Center and Clinica K-3) in Matamoros, Mexico, during January 1-May 13, 2023. This report describes outbreak epidemiology and outlines interim diagnostic and treatment recommendations. METHODS: Interim recommendations for diagnosis and management were developed by the Mycoses Study Group Research Education and Consortium (MSGERC) based on the clinical experience of clinicians caring for patients during the current outbreak or during previous outbreaks of healthcare-associated fungal meningitis in Durango, Mexico, and the United States. RESULTS: As of July 7, 2023, the situation has evolved into a multistate and multinational fungal meningitis outbreak. A total of 185 residents in 22 U.S. states and jurisdictions have been identified who might be at risk of fungal meningitis because they received epidural anesthesia at the clinics of interest in 2023. Among these patients, 11 suspected, 10 probable, and 10 confirmed U.S. cases have been diagnosed, with severe vascular complications and eight deaths occurring. Fusarium solani species complex has been identified as the causative agent, with antifungal susceptibility testing of a single isolate demonstrating poor in vitro activity for most available antifungals. Currently, triple therapy with intravenous voriconazole, liposomal amphotericin B, and fosmanogepix is recommended. CONCLUSIONS: Efforts to understand the source of this outbreak and optimal treatment approaches are ongoing, but infectious diseases physicians should be aware of available treatment recommendations. New information will be available on CDC's website.

Kneiffiella palmae: A non-Aspergillus fungal infection isolated from a pulmonary nodule in a child with chronic granulomatous disease.

We report the first known human case of Kneiffiella palmae in the medical literature. K. palmae was isolated from a pulmonary nodule in a 7-year-old male with chronic granulomatous disease. The mold was identified as K. palmae at a national reference laboratory, where 17 other human respiratory samples tested positive for K. palmae from 2013 to 2021. Optimal antimicrobial treatment is unknown, but azoles and amphotericin B demonstrated in vitro activity against each tested isolate.

Low Rates of Antifungal Therapeutic Drug Monitoring Among Inpatients Who Received Itraconazole, Posaconazole, or Voriconazole, United States, 2019-2021.

Antifungal therapeutic drug monitoring (TDM) is recommended for hospitalized patients receiving itraconazole, posaconazole, or voriconazole for treatment or prophylaxis. In this analysis of hospital-based data, TDM was uncommonly performed (15.8%) in a large cohort of eligible patients, suggesting missed opportunities to avoid subtherapeutic drug levels and minimize toxicity.

Terbinafine-Resistant Dermatophytes and the Presence of Trichophyton indotineae in North America.

Dermatophytes are common causes of skin, hair, and nail infections in humans. The most common species causing infections in humans are Trichophyton rubrum, Trichophyton mentagrophytes, and Trichophyton interdigitale. Outbreaks of recalcitrant dermatophytosis have been reported in parts of South Asia, including those caused by a hypervirulent and resistant species, Trichophyton indotineae. We evaluated the antifungal susceptibility profiles of dermatophytes received by our laboratory from institutions across North America between 2021 and 2022 and performed species identification for isolates deemed to demonstrate in vitro resistance. Susceptibility testing was performed by CLSI broth microdilution methods, and species identification was performed by DNA sequence analysis. During this 2-year period, 271 dermatophyte isolates were included, the majority of which demonstrated low MIC values for terbinafine (geometric mean [GM] and modal MIC, 0.031 µg/mL and 0.008 µg/mL, respectively) and the azoles itraconazole, posaconazole, and voriconazole (0.035 to 0.049 µg/mL and ≤0.03 µg/mL). However, 18.6% of the isolates tested were resistant to terbinafine (MIC ≥ 0.5 µg/mL), including 21 T. rubrum and 21 T. indotineae isolates. These isolates were received from several different states in the United States and two provinces in Canada. In contrast, resistance to itraconazole was relatively rare. We also searched our laboratory database for earlier isolates that were resistant to terbinafine and identified 3 additional T. indotineae isolates, the earliest of which was from 2017. These results demonstrate that terbinafine resistance in dermatophytes was relatively common over this 2-year period and that T. indotineae is present in multiple areas in North America. Continued surveillance is warranted.

Utility of triazole antifungal therapeutic drug monitoring: Insights from the Society of Infectious Diseases Pharmacists: Endorsed by the Mycoses Study Group Education and Research Consortium.

Triazole antifungals (i.e., fluconazole, itraconazole, voriconazole, posaconazole, and isavuconazole) are commonly used in clinical practice to prevent or treat invasive fungal infections. Most triazole antifungals require therapeutic drug monitoring (TDM) due to highly variable pharmacokinetics, known drug interactions, and established relationships between exposure and response. On behalf of the Society of Infectious Diseases Pharmacists (SIDP), this insight describes the pharmacokinetic principles and pharmacodynamic targets of commonly used triazole antifungals and provides the rationale for utility of TDM within each agent.

Screening the medicine for malaria venture's Pandemic Response Box to identify novel inhibitors of Candida albicans and Candida auris biofilm formation.

Candida spp. are opportunistic yeasts capable of forming biofilms, which contribute to resistance, increasing the urgency for new effective antifungal therapies. Repurposing existing drugs could significantly accelerate the development of novel therapies against candidiasis. We screened the Pandemic Response Box containing 400 diverse drug-like molecules active against bacteria, viruses or fungi, for inhibitors of Candida albicans and Candida auris biofilm formation. Initial hits were identified based on the demonstration of >70% inhibitory activity. Dose-response assays were used to confirm the antifungal activity of initial hits and establish their potency. The spectrum of antifungal activity of the leading compounds was determined against a panel of medically important fungi, and the in vivo activity of the leading repositionable agent was evaluated in murine models of C. albicans and C. auris systemic candidiasis. The primary screening identified 20 hit compounds, and their antifungal activity and potency against C. albicans and C. auris were validated using dose-response measurements. From these experiments, the rapalog everolimus, emerged as the leading repositionable candidate. Everolimus displayed potent antifungal activity against different Candida spp., but more moderate levels of activity against filamentous fungi. Treatment with everolimus increased survival of mice infected with C. albicans, but not those with C. auris. The screening of the Pandemic Response Box resulted in the identification of several drugs with novel antifungal activity, with everolimus emerging as the main repositionable candidate. Further in vitro and in vivo studies are needed to confirm its potential therapeutic use.

Systemic Exophiala equina infection in an Eastern box turtle (Terrapene carolina carolina): a case report and literature review.

Phaeohyphomycosis is an infection caused by melanized fungi. This disease has been reported in several animal species including invertebrates, cold-blooded vertebrates, mammals, and humans. Melanized fungi have similar phenotypical features and confirmation requires culture and molecular diagnostics. To exemplify this we present a case of a 333 g adult of unknown age, free-ranging, male Eastern box turtle (Terrapene carolina carolina) that was referred to the Turtle Rescue Team at North Carolina State University for evaluation of multilobulated masses occupying the entire left orbit and at the right forelimb on the plantarolateral aspect of the foot. A fine needle aspirate cytologic examination of the mass on the right forelimb revealed large numbers of inflammatory cells and fungal organisms. Histopathology of the skin biopsies from the right forefoot was consistent with phaeohyphomycosis. A course of antifungal medication was started (Fluconazole 21 mg/kg loading dose IV then 5 mg/kg PO SID q 30 days). Due to concern for the patient's quality of life and the lack of a curative treatment plan, humane euthanasia was elected. Gross and histological postmortem examination confirmed the presence of multiple coelomic masses similar in appearance to those observed in the left orbit and right forefoot indicating disseminated phaeohyphomycosis. A swab of the periocular mass was submitted for fungal culture and phenotypic identification. The isolate was later identified as Exophiala equina through a combination of phenotypic characterization and sequencing of the ITS region of the nuclear rDNA. Exophiala is a genus in the family Herpotrichiellaceae, order Chaetothyriales and is considered an opportunistic "black yeast" causing infection in aquatic invertebrates, fish, amphibians, reptiles, and mammals including humans. Exophiala equina is infrequently reported in animals, with only three cases in the literature including the herein report.

hapE and hmg1 Mutations Are Drivers of cyp51A-Independent Pan-Triazole Resistance in an Aspergillus fumigatus Clinical Isolate.

Aspergillus fumigatus is a ubiquitous environmental mold that can cause severe disease in immunocompromised patients and chronic disease in individuals with underlying lung conditions. Triazoles are the most widely used class of antifungal drugs to treat A. fumigatus infections, but their use in the clinic is threatened by the emergence of triazole-resistant isolates worldwide, reinforcing the need for a better understanding of resistance mechanisms. The predominant mechanisms of A. fumigatus triazole resistance involve mutations affecting the promoter region or coding sequence of the target enzyme of the triazoles, Cyp51A. However, triazole-resistant isolates without cyp51A-associated mutations are frequently identified. In this study, we investigate a pan-triazole-resistant clinical isolate, DI15-105, that simultaneously carries the mutations hapEP88L and hmg1F262del, with no mutations in cyp51A. Using a Cas9-mediated gene-editing system, hapEP88L and hmg1F262del mutations were reverted in DI15-105. Here, we show that the combination of these mutations accounts for pan-triazole resistance in DI15-105. To our knowledge, DI15-105 is the first clinical isolate reported to simultaneously carry mutations in hapE and hmg1 and only the second with the hapEP88L mutation. IMPORTANCE Triazole resistance is an important cause of treatment failure and high mortality rates for A. fumigatus human infections. Although Cyp51A-associated mutations are frequently identified as the cause of A. fumigatus triazole resistance, they do not explain the resistance phenotypes for several isolates. In this study, we demonstrate that hapE and hmg1 mutations additively contribute to pan-triazole resistance in an A. fumigatus clinical isolate lacking cyp51-associated mutations. Our results exemplify the importance of and the need for a better understanding of cyp51A-independent triazole resistance mechanisms.

Fungal keratitis caused by Coniochaeta mutabilis-A case report.

We present a rare case of recalcitrant fungal keratitis caused by Coniochaeta mutabilis, successfully managed with a course of oral, topical, intrastromal, and intracameral antifungals. A 57-year-old male on their fourth week of treatment for presumed left herpes simplex keratitis presented to clinic with severe left-sided foreign body sensation after gardening in his yard. On examination, a white corneal plaque was observed at 8 o'clock, shown to be a dense collection of fungal hyphae on confocal microscopy. Corneal cultures revealed yeast-like cells, initially identified as Kabatiella zeae by matching 100% identity with K. zeae strains CBS 767.71 and CBS 265.32 in BLASTn search using the internal transcribed spacer (ITS) sequence. Treated for over four months with topical amphotericin B and oral voriconazole without improvement, recourse to intrastromal and intracameral amphotericin B injections, coupled with the application of cyanoacrylate glue to the lesion and a bandage contact lens, led to eventual resolution. The patient subsequently underwent cataract surgery, achieving a BCVA of 20/20 in the eye. Surprisingly, upon further sequence analyses of combined ITS and large subunit ribosomal ribonucleic acid (LSU) and investigation of the K. zeae German strain CBS 767.71, the organism was revealed to be Coniochaeta mutabilis (formerly Lecythospora mutabilis). This means that the correct name for CBS 767.71 and CBS 265.32 is C. mutabilis and should be corrected in the GenBank record to avoid misleading identification in the future. This case also underscores the urgent unmet need for improved molecular diagnostic modalities in the care of corneal infections.

Antifungal Susceptibility of Yeasts and Filamentous Fungi by CLSI Broth Microdilution Testing.

Antifungal susceptibility testing is performed against yeasts and filamentous fungi (molds) for various purposes. In clinical settings, the results of these in vitro assays may be used to help guide therapy for individual patients. Surveillance studies use susceptibility testing to monitor for the development of resistance to different agents. These assays are also frequently used in preclinical drug development to determine the spectrum of activity and assess the in vitro potency of investigational agents. Broth microdilution is a form of antifungal susceptibility testing that is frequently used for each of these purposes. The Clinical and Laboratory Standards Institute (CLSI) has developed standardized methods for broth microdilution antifungal susceptibility testing of both yeasts and molds. These methods are useful in the clinical diagnostics arena for determining if a particular fungal isolate may be resistant to clinically available antifungals, and they are also amendable for research purposes, as the microtiter trays can be prepared in different formats with different antifungal and investigational agent concentration ranges and with different growth media if necessary. This chapter describes the CLSI methods for antifungal susceptibility testing by broth microdilution for both yeasts and molds, how endpoints are read for different classes of antifungal agents, and how the results may be interpreted.

Species Distribution and Antifungal Susceptibilities of Aspergillus Section Terrei Isolates in Clinical Samples from the United States and Description of Aspergillus pseudoalabamensis sp. nov.

Aspergillus section Terrei consists of numerous cryptic species in addition to A. terreus sensu stricto. The treatment of invasive infections caused by these fungi may pose a unique challenge prior to diagnosis and species identification, in that they are often clinically resistant to amphotericin B, with poor outcomes and low survival rates in patients treated with this polyene. Data on the species distributions and susceptibility profiles of isolates within section Terrei from the United States (U.S.) are limited. Here, we report the species distributions and susceptibility profiles for amphotericin B, isavuconazole, itraconazole, posaconazole, voriconazole, and micafungin against 278 clinical isolates of this section from institutions across the U.S. collected over a 52-month period. Species identification was performed by DNA sequence analysis and phenotypic characterization. Susceptibility testing was performed using the CLSI broth microdilution method. The majority of isolates were identified as Aspergillus terreus sensu stricto (69.8%), although several other cryptic species were also identified. Most were cultured from specimens collected from the respiratory tract. Posaconazole demonstrated the most potent activity of the azoles (MIC range ≤ 0.03-1 mg/L), followed by itraconazole (≤0.03-2 mg/L), voriconazole, and isavuconazole (0.125-8 mg/L for each). Amphotericin B demonstrated reduced in vitro susceptibility against this section (MIC range 0.25-8 mg/L), although this appeared to be species-dependent. A new species within this section, A. pseudoalabamensis, is also described. Our results, which are specific to the U.S., are similar to previous surveillance studies of the Aspergillus section Terrei.

Phialophora chinensis fungal keratitis: An initial case report and species identification.

Purpose: To report the initial case of microbial keratitis caused by Phialophora chinensis, a rare cause of fungal keratitis. Observations: A 66-year-old gentleman with a complex right eye (OD) ocular history including herpes simplex virus infectious epithelial keratitis with subsequent neurotrophic keratopathy, and prior combined Candida albicans and parapsilosis fungal keratitis presented with pain OD in the absence of an antecedent trauma. The patient was found to have a filamentous fungal keratitis, which was subsequently cultured and identified as Phialophora chinensis by the laboratory. Despite topical and oral antifungal treatment based on sensitivities determined by the lab, the patient ultimately required intrastromal and subconjunctival antifungal injections, corneal crosslinking, and superficial keratectomy with amniotic membrane to clinically improve. The fungal keratitis recurred twice, with each occurrence rapidly progressing to corneal perforation. Months after the second penetrating keratoplasty, the patient's mental status declined due to multiorgan failure. An occult pulmonary malignancy was discovered during this hospital stay, and the patient was lost to follow-up after entering hospice. Conclusions and Importance: We report a unique case of fungal keratitis caused by Phialophora chinensis and the subsequent management, including both medical and surgical interventions. Despite a multimodal treatment regimen, this case demonstrates the recalcitrant and potentially recurrent nature of fungal keratitis caused by P. chinensis.

Dihydroorotate dehydrogenase inhibitor olorofim has potent in vitro activity against Microascus/Scopulariopsis, Rasamsonia, Penicillium and Talaromyces species.

BACKGROUND: Treatment options against infections caused by rare but emerging moulds may be limited by their reduced susceptibility or resistance to clinically available antifungals. The investigational antifungal olorofim, which targets the biosynthesis of pyrimidines within fungi, has activity against different species of filamentous fungi, including Aspergillus and Scedosporium/Lomentospora prolificans isolates that are resistant to available antifungals. OBJECTIVE: We evaluated the in vitro activity of olorofim against 160 isolates within the genera Microascus/Scopulariopsis, Penicillium, Talaromyces and the Rasamsonia argillacea species complex. METHODS: One hundred sixty clinical isolates that had previously been identified to the species level by DNA sequence analysis were included. Antifungal susceptibility testing was performed by CLSI M38 broth microdilution for olorofim, amphotericin B, caspofungin, posaconazole and voriconazole. RESULTS: Olorofim demonstrated in vitro activity against each of the genera tested. Overall, olorofim MICs ranged from ≤0.008 to 0.5 mg/L against all isolates tested, with MIC90 and modal MIC values ranging from ≤0.008 to 0.25 mg/L and ≤0.008 to 0.03 mg/L, respectively. This activity was also maintained against individual isolates that had reduced susceptibility to or in vitro resistance against amphotericin B, posaconazole and/or voriconazole. CONCLUSIONS: The investigational agent olorofim demonstrated good in vitro activity against clinical isolates of emerging mould pathogens, including those with reduced susceptibility or resistance to clinically available antifungals. Further studies are warranted to determine how well this in vitro activity translates into in vivo efficacy against infections caused by these fungi.