Suillus: an emerging model for the study of ectomycorrhizal ecology and evolution.

Research on mycorrhizal symbiosis has been slowed by a lack of established study systems. To address this challenge, we have been developing Suillus, a widespread ecologically and economically relevant fungal genus primarily associated with the plant family Pinaceae, into a model system for studying ectomycorrhizal (ECM) associations. Over the last decade, we have compiled extensive genomic resources, culture libraries, a phenotype database, and protocols for manipulating Suillus fungi with and without their tree partners. Our efforts have already resulted in a large number of publicly available genomes, transcriptomes, and respective annotations, as well as advances in our understanding of mycorrhizal partner specificity and host communication, fungal and plant nutrition, environmental adaptation, soil nutrient cycling, interspecific competition, and biological invasions. Here, we highlight the most significant recent findings enabled by Suillus, present a suite of protocols for working with the genus, and discuss how Suillus is emerging as an important model to elucidate the ecology and evolution of ECM interactions.

SWI/SNF complex-mediated chromatin remodeling in Candida glabrata promotes immune evasion.

Immune evasion is critical for fungal virulence. However, how the human opportunistic pathogen Candida glabrata (Cg) accomplishes this is unknown. Here, we present the first genome-wide nucleosome map of the macrophage-internalized Cg consisting of ∼12,000 dynamic and 70,000 total nucleosomes. We demonstrate that CgSnf2 (SWI/SNF chromatin remodeling complex-ATPase subunit)-mediated chromatin reorganization in macrophage-internalized Cg upregulates and downregulates the immunosuppressive seven-gene mannosyltransferase-cluster (CgMT-C) and immunostimulatory cell surface adhesin-encoding EPA1 gene, respectively. Consistently, EPA1 overexpression and CgMT-C deletion elevated IL-1ß (pro-inflammatory cytokine) production and diminished Cg proliferation in macrophages. Further, Cgsnf2Δ had higher Epa1 surface expression, and evoked increased IL-1ß secretion, and was killed in macrophages. Akt-, p38-, NF-κB- or NLRP3 inflammasome-inhibition partially reversed increased IL-1ß secretion in Cgsnf2Δ-infected macrophages. Importantly, macrophages responded to multiple Candida pathogens via NF-κB-dependent IL-1ß production, underscoring NF-κB signaling's role in fungal diseases. Altogether, our findings directly link the nucleosome positioning-based chromatin remodeling to fungal immunomodulatory molecule expression.

Biogenesis, germination, and pathogenesis of Cryptococcus spores.

SUMMARYSpores are primary infectious propagules for the majority of human fungal pathogens; however, relatively little is known about their fundamental biology. One strategy to address this deficiency has been to develop the basidiospores of Cryptococcus into a model for pathogenic spore biology. Here, we provide an update on the state of the field with a comprehensive review of the data generated from the study of Cryptococcus basidiospores from their formation (sporulation) and differentiation (germination) to their roles in pathogenesis. Importantly, we provide support for the presence of basidiospores in nature, define the key characteristics that distinguish basidiospores from yeast cells, and clarify their likely roles as infectious particles. This review is intended to demonstrate the importance of basidiospores in the field of Cryptococcus research and provide a solid foundation from which researchers who wish to study sexual spores in any fungal system can launch their studies.

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.

Resistance Profile, Terbinafine Resistance Screening and MALDI-TOF MS Identification of the Emerging Pathogen Trichophyton indotineae.

The emerging pathogen Trichophyton indotineae, often resistant to terbinafine (TRB), is known to cause severe dermatophytoses such as tinea corporis and tinea cruris. In order to achieve successful treatment for these infections, insight in the resistance profile of T. indotineae strains and rapid, reliable identification is necessary. In this research, a screening medium was tested on T. indotineae strains (n = 20) as an indication tool of TRB resistance. The obtained results were confirmed by antifungal susceptibility testing (AST) for TRB following the in vitro broth microdilution reference method. Additionally, AST was performed for eight other antifungal drugs: fluconazole, itraconazole, voriconazole, ketoconazole, griseofulvin, ciclopirox olamine, naftifine and amorolfine. Forty-five percent of the strains were confirmed to be resistant to terbinafine. The TRB resistant strains showed elevated minimal inhibitory concentration values for naftifine and amorolfine as well. DNA sequencing of the squalene epoxidase-encoding gene showed that TRB resistance was a consequence of missense point mutations in this gene, which led to amino acid substitutions F397L or L393F. MALDI-TOF MS was used as a quick, accurate identification tool for T. indotineae, as it can be challenging to distinguish it from closely related species such as Trichophyton mentagrophytes or Trichophyton interdigitale using morphological characteristics. While MALDI-TOF MS could reliably identify ≥ 95% of the T. indotineae strains (depending on the spectral library), it could not be used to successfully distinguish TRB susceptible from TRB resistant strains.

Exploring fungal diversity in Vietnam: A citizen science initiative.

Invasive fungal diseases are increasing globally, causing a large burden of disease in vulnerable populations. At the same time, antifungal resistance is rapidly emerging. Affordable nationwide and regional surveillance of fungal pathogens is needed. We have adapted a citizen-science methodology developed by a United Kingdom research group to study six key fungi in Vietnam, where there is no existing formal surveillance. These pathogens were ranked as high or critical in the World Health Organization fungal priority pathogens list and recognized as major disease-causing agents in Vietnam. Secondary school students (n = 90) in Hanoi were our citizen scientists, collecting soil (n = 90) and air (n = 90) samples for fungal identification and characterisation of drug-susceptibility in the laboratory. Pilot studies confirmed the effectiveness of our revised isolation procedure, which used selective culture media to improve the isolation of target fungi. Through active school and student involvement, optimized protocols, and our cost-effective sampling, the study could be scaled across Vietnam. We demonstrate an approach to fungal surveillance which also enhances science education, and awareness of fungal diseases. It addresses critical healthcare and education challenges in Vietnam while combating the growing issues of invasive fungal diseases and antifungal resistance.

A Case of Disseminated Histoplasmosis Presenting in a 65-Year-Old Male Without Apparent Immunodeficiency Successfully Treated With Isavuconazole.

Histoplasma capsulatum causes symptoms in fewer than 5% of infected people, with most recovering without treatment two to three weeks after the onset of symptoms. Progressive disseminated histoplasmosis in adults occurs most often in persons with underlying immunodeficiency. We present a case of a 65-year-old caucasian male without any known immune defect from North Alabama, United States, presenting with chronic tongue ulcer and constitutional symptoms. CT and positron emission tomography scans showed disseminated infection with pulmonary nodule, oral/buccal lesions, and bilateral adrenal hyperplasia. The patient's left adrenal gland and tongue were biopsied and stains confirmed the presence of histoplasmosis in both samples. The patient was treated with isavuconazole off-label as per the United States FDA. The patient tolerated the therapy well and had symptomatic improvement. A follow-up CT scan showed improvement and resolution of adrenal masses.

The Cologne ECMM Excellence Center: A Two-Year Analysis of External Consultation Service for Invasive Fungal Infections.

The European Confederation of Medical Mycology (ECMM), formed due to the surge in invasive fungal infections (IFI), initiated the Excellence Centers program in 2016 to guide stakeholders to leading medical mycology sites. This report focuses on the Cologne ECMM Excellence Center, recognized with Diamond status for active global involvement in 2017. The center offers free consultation via email and phone, responding within 24 h for life-threatening IFI, collecting data on origin, pathogens, infection details, and more. Over two years, 189 requests were received globally, predominantly from Germany (85%), mainly involving Aspergillus spp., Mucorales, and Candida spp. Fungal mixed infections occurred in 4% of cases. The center's service effectively addresses IFI challenges, advocating for a comprehensive study encompassing all ECMM Excellence Centers to enhance global mycological care. Proactive expansion of consultancy platforms is crucial, with future analyses needed to assess expert advice's impact on patient outcomes.

Pan-Indian Clinical Registry of Invasive Fungal Infections Among Patients in the Intensive Care Unit: Protocol for a Multicentric Prospective Study.

BACKGROUND: Fungal infections are now a great public health threat, especially in those with underlying risk factors such as neutropenia, diabetes, high-dose steroid treatment, cancer chemotherapy, prolonged intensive care unit stay, and so on, which can lead to mycoses with higher mortality rates. The rates of these infections have been steadily increasing over the past 2 decades due to the increasing population of patients who are immunocompromised. However, the data regarding the exact burden of such infection are still not available from India. Therefore, this registry was initiated to collate systematic data on invasive fungal infections (IFIs) across the country. OBJECTIVE: The primary aim of this study is to create a multicenter digital clinical registry and monitor trends of IFIs and emerging fungal diseases, as well as early signals of any potential fungal outbreak in any region. The registry will also capture information on the antifungal resistance patterns and the contribution of fungal infections on overall morbidity and inpatient mortality across various conditions. METHODS: This multicenter, prospective, noninterventional observational study will be conducted by the Indian Council of Medical Research through a web-based data collection method from 8 Advanced Mycology Diagnostic and Research Centers across the country. Data on age, gender, clinical signs and symptoms, date of admission, date of discharge or death, diagnostic tests performed, identified pathogen details, antifungal susceptibility testing, outcome, and so on will be obtained from hospital records. Descriptive and multivariate statistical methods will be applied to investigate clinical manifestations, risk variables, and treatment outcomes. RESULTS: These Advanced Mycology Diagnostic and Research Centers are expected to find the hidden cases of fungal infections in the intensive care unit setting. The study will facilitate the enhancement of the precision of fungal infection diagnosis and prompt treatment modalities in response to antifungal drug sensitivity tests. This registry will improve our understanding of IFIs, support evidence-based clinical decision-making ability, and encourage public health policies and actions. CONCLUSIONS: Fungal diseases are a neglected public health problem. Fewer diagnostic facilities, scanty published data, and increased vulnerable patient groups make the situation worse. This is the first systematic clinical registry of IFIs in India. Data generated from this registry will increase our understanding related to the diagnosis, treatment, and prevention of fungal diseases in India by addressing pertinent gaps in mycology. This initiative will ensure a visible impact on public health in the country. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/54672.

Slicing through the challenge of maintaining Pneumocystis in the laboratory.

Pneumocystis jirovecii is a major fungal pathogen of humans that causes life-threatening lung infections in immunocompromised individuals. Despite its huge global impact upon human health, our understanding of the pathobiology of this deadly fungus remains extremely limited, largely because it is not yet possible to cultivate Pneumocystis in vitro, independently of the host. However, a recent paper by Munyonho et al. offers a major step forward (F. T. Munyonho, R. D. Clark, D. Lin, M. S. Khatun, et al., 2023, mBio 15:e01464-23, https://doi.org/10.1128/mbio.01464-23). They show that it is possible to maintain both the trophozoite and cyst forms of the mouse pathogen, Pneumocystis murina, in precision-cut lung slices for several weeks. Furthermore, they demonstrate that this offers the exciting opportunity to examine potential virulence factors such as possible biofilm formation as well as antifungal drug responses in the lung.

Targeting host tyrosine kinase receptor EphA2 signaling via small-molecule ALW-II-41-27 inhibits macrophage pro-inflammatory signaling responses to Pneumocystis carinii ß-glucans.

Pneumocystis jirovecii, the fungus that causes Pneumocystis jirovecii pneumonia (PJP), is a leading cause of morbidity and mortality in immunocompromised individuals. We have previously shown that lung epithelial cells can bind Pneumocystis spp. ß-glucans via the EphA2 receptor, resulting in activation and release of proinflammatory cytokines. Herein, we show that in vivo Pneumocystis spp. ß-glucans activation of the inflammatory signaling cascade in macrophages can be pharmacodynamically inhibited with the EphA2 receptor small-molecule inhibitor ALW-II-41-27. In vitro, when ALW-II-41-27 is administrated via intraperitoneal to mice prior to the administration of highly proinflammatory Saccharomyces cerevisiae ß-glucans in the lung, a significant reduction in TNF-alpha release was noted in the ALW-II-41-27 pre-treated group. Taken together, our data suggest that targeting host lung macrophage activation via EphA2 receptor-fungal ß-glucans interactions with ALW-II-41-27 or other EphA2 receptor kinase targeting inhibitors might be an attractive and viable strategy to reduce detrimental lung inflammation associated with PJP.

Sac1 links phosphoinositide turnover to cryptococcal virulence.

Cryptococcus neoformans is an environmentally-acquired fungal pathogen that causes over 140,000 deaths per year. Cryptococcal infection occurs when infectious particles are deposited into the lung, where they encounter host phagocytic cells. C. neoformans may be engulfed by these phagocytes, an important step of infection that leads to out-comes ranging from termination of infection to cryptococcal dissemination. To study this critical process, we screened approximately 4,700 cryptococcal gene deletion mutants for altered uptake, using primary mouse and human phagocytic cells. Among the hits of this screen, we identified 93 mutants with perturbed uptake in both systems, as well as others with differences in uptake by only one cell type. We further screened the hits for changes in thickness of the capsule, a protective polysaccharide layer around the cell which is an important cryptococcal virulence factor. This second screen yielded 131 mutants, including one lacking the phosphatidylinositol-4-phosphate phosphatase Sac1. In this work, we implicate Sac1 in both host cell uptake and capsule production. We found that sac1 mutants exhibit lipid trafficking defects, reductions in secretory system function, and changes in capsule size and composition. Many of these changes occur specifically in tissue culture media, highlighting the role of Sac1 phosphatase activity in responding to the stress of host-like conditions. Overall, these findings show how genome-scale screening can identify cellular factors that contribute to our understanding of cryptococcal biology and demonstrate the role of Sac1 in determining fungal virulence.

The Development of a Clinical Registry Digital Database on Invasive Fungal Infections in India: Advancing Epidemiological Understanding and Patient Care.

A well-structured digital database is essential for any national priority project as it can provide real-time data analysis and facilitate quick decision making. In recent times, particularly after the COVID-19 pandemic, invasive fungal infections (IFIs) have emerged as a significant public health challenge in India, affecting vulnerable population, including immunocompromised individuals. The lack of comprehensive and well-structured data on IFIs has hindered efforts to understand their true burden and optimize patient care. To address this critical knowledge gap, the ICMR has undertaken a Pan-India pioneer initiative to develop a network of Advanced Mycology Diagnostic research centres in different geographical zones of the country (ICMR-MycoNet). Under the aegis of this project, a clinical registry on IFIs in the ICUs is initiated. This process paper presents a detailed account of the steps involved in the establishment of a web-based data entering and monitoring platform to capture data electronically, ensuring robust and secure data collection and management. This system not only allows participating ICMR-MycoNet centres to enter patient information directly into the database using standardized Case Report Form (CRF) but also includes data validation checks to ensure the accuracy and completeness of entered data. It is complemented by a real-time, web-based, and adaptable data visualization platform. This registry aims to provide crucial epidemiological insights, promote evidence-based hospital infection control programs, and ultimately improve patient outcomes in the face of this formidable healthcare challenge.

Coccidioidomycosis presenting years after returning from travel.

After having traveled to California in 2017, a 26-year old Dutch man presented in 2020 with persisting cough and shortness of breath. Radiology showed cystic bronchiectasis with peri-bronchial consolidation in his right upper lobe. Laboratory studies in August 2021 showed an increased total IgE, specific Aspergillus IgE, eosinophilia and positive BAL culture for Coccidioides immitis/posadasii. After 6 weeks of itraconazole treatment for suspected allergic bronchopulmonary aspergillosis, symptoms persisted and respiratory cultures remained positive. The infection was cleared after a 6-month course of fluconazole. (max 75 words).

The mutual value of histopathology and ITS sequencing in the diagnosis of mucormycosis.

AIMS: Mucormycosis is a fast-progressing disease with a high mortality rate. The most important factor determining survival of patients is early and accurate diagnosis. Although histopathology often recognises invasive mould infections at first, histomorphology alone is insufficient in providing an accurate diagnosis. Unbiased molecular methods to detect and identify fungi are promising, yet their role in complementing routine histopathological workflows has not been studied sufficiently. METHODS AND RESULTS: We performed a retrospective single-centre study examining the clinical value of complementing histopathology with internal transcribed spacer (ITS) sequencing of fungal DNA in the routine diagnosis of mucormycosis. At our academic centre, we identified 14 consecutive mucormycosis cases diagnosed by histopathology and subsequent ITS sequencing. Using histomorphological examination, fungal hyphae could be detected in all cases; however, morphological features were unreliable regarding specifying the taxa. Subsequent ITS sequencing identified a remarkable phylogenetic diversity among Mucorales: the most common species was Rhizopus microsporus (six of 14; 42.9%), followed by Lichtheimia corymbifera (three of 14, 21.4%) and single detections of Rhizopus oryzae, Actinomucor elegans, Mucor circinelloides, Rhizomucor pusillus and Rhizomucor miehei (one of 14; 7.1%, respectively). In one case, we additionally detected Pneumocystis jirovecii in the same lung tissue specimen, suggesting a clinically relevant co-infection. Fungal culture was performed in 10 cases but yielded positive results in only two of 10 (20%), revealing its limited value in the diagnosis of mucormycosis. CONCLUSIONS: Our study demonstrates that a combination of histopathology and ITS sequencing is a practically feasible approach that outperforms fungal culture in detecting Mucorales in tissue-associated infections. Therefore, pathologists might adapt diagnostic workflows accordingly when mucormycosis is suspected.

Multicentric Study on the Clinical Mycology Capacity and Access to Antifungal Treatment in Portugal.

The success of the clinical management of invasive fungal diseases (IFD) is highly dependent on suitable tools for timely and accurate diagnosis for effective treatment. An in-depth analysis of the ability of European institutions to promptly and accurately diagnose IFD was previously conducted to identify limitations and aspects to improve. Here, we evaluated and discussed the specific case of Portugal, for which, to our knowledge, there are no reports describing the national mycological diagnostic capacity and access to antifungal treatment. Data from 16 Portuguese medical institutions were collected via an online electronic case report form covering different parameters, including institution profile, self-perceived IFD incidence, target patients, diagnostic methods and reagents, and available antifungals. The majority of participating institutions (69%) reported a low-very low incidence of IFD, with Candida spp. indicated as the most relevant fungal pathogen, followed by Aspergillus spp. and Cryptococcus spp. All institutions had access to culture and microscopy, whereas 94 and 88% were able to run antigen-detection assays and molecular tests, respectively. All of the institutions capable of providing antifungal therapy declared to have access to at least one antifungal. However, echinocandins were only available at 85% of the sites. Therapeutic drug monitoring (TDM) was reported to remain a very restricted practice in Portugal, being available in 19% of the institutions, with the TDM of itraconazole and posaconazole performed in only 6% of them. Importantly, several of these resources are outsourced to external entities. Except for TDM, Portugal appears to be well-prepared concerning the overall capacity to diagnose and treat IFD. Future efforts should focus on promoting the widespread availability of TDM and improved access to multiple classes of antifungals, to further improve patient outcomes.

The ATCC genome portal: 3,938 authenticated microbial reference genomes.

The ATCC Genome Portal (AGP, https://genomes.atcc.org/) is a database of authenticated genomes for bacteria, fungi, protists, and viruses held in ATCC's biorepository. It now includes 3,938 assemblies (253% increase) produced under ISO 9000 by ATCC. Here, we present new features and content added to the AGP for the research community.