Human gut mycobiota tune immunity via CARD9-dependent induction of anti-fungal IgG antibodies.

Antibodies mediate natural and vaccine-induced immunity against viral and bacterial pathogens, whereas fungi represent a widespread kingdom of pathogenic species for which neither vaccine nor neutralizing antibody therapies are clinically available. Here, using a multi-kingdom antibody profiling (multiKAP) approach, we explore the human antibody repertoires against gut commensal fungi (mycobiota). We identify species preferentially targeted by systemic antibodies in humans, with Candida albicans being the major inducer of antifungal immunoglobulin G (IgG). Fungal colonization of the gut induces germinal center (GC)-dependent B cell expansion in extraintestinal lymphoid tissues and generates systemic antibodies that confer protection against disseminated C. albicans or C. auris infection. Antifungal IgG production depends on the innate immunity regulator CARD9 and CARD9+CX3CR1+ macrophages. In individuals with invasive candidiasis, loss-of-function mutations in CARD9 are associated with impaired antifungal IgG responses. These results reveal an important role of gut commensal fungi in shaping the human antibody repertoire through CARD9-dependent induction of host-protective antifungal IgG.

Harnessing the Immune Response to Fungal Pathogens for Vaccine Development.

Invasive fungal infections are emerging diseases that kill over 1.5 million people per year worldwide. With the increase of immunocompromised populations, the incidence of invasive fungal infections is expected to continue to rise. Vaccines for viral and bacterial infectious diseases have had a transformative impact on human health worldwide. However, no fungal vaccines are currently in clinical use. Recently, interest in fungal vaccines has grown significantly. One Candida vaccine has completed phase 2 clinical trials, and research on vaccines against coccidioidomycosis continues to advance. Additionally, multiple groups have discovered various Cryptococcus mutant strains that promote protective responses to subsequent challenge in mouse models. There has also been progress in antibody-mediated fungal vaccines. In this review, we highlight recent fungal vaccine research progress, outline the wealth of data generated, and summarize current research for both fungal biology and immunology studies relevant to fungal vaccine development. We also review technological advancements in vaccine development and highlight the future prospects of a human vaccine against invasive fungal infections.

Exploiting antifungal immunity in the clinical context.

The continuous expansion of immunocompromised patient populations at-risk for developing life-threatening opportunistic fungal infections in recent decades has helped develop a deeper understanding of antifungal host defenses, which has provided the foundation for eventually devising immune-based targeted interventions in the clinic. This review outlines how genetic variation in certain immune pathway-related genes may contribute to the observed clinical variability in the risk of acquisition and/or severity of fungal infections and how immunogenetic-based patient stratification may enable the eventual development of personalized strategies for antifungal prophylaxis and/or vaccination. Moreover, this review synthesizes the emerging cytokine-based, cell-based, and other immunotherapeutic strategies that have shown promise as adjunctive therapies for boosting or modulating tissue-specific antifungal immune responses in the context of opportunistic fungal infections.

The Toll pathway mediates Drosophila resilience to Aspergillus mycotoxins through specific Bomanins.

Host defense against infections encompasses both resistance, which targets microorganisms for neutralization or elimination, and resilience/disease tolerance, which allows the host to withstand/tolerate pathogens and repair damages. In Drosophila, the Toll signaling pathway is thought to mediate resistance against fungal infections by regulating the secretion of antimicrobial peptides, potentially including Bomanins. We find that Aspergillus fumigatus kills Drosophila Toll pathway mutants without invasion because its dissemination is blocked by melanization, suggesting a role for Toll in host defense distinct from resistance. We report that mutants affecting the Toll pathway or the 55C Bomanin locus are susceptible to the injection of two Aspergillus mycotoxins, restrictocin and verruculogen. The vulnerability of 55C deletion mutants to these mycotoxins is rescued by the overexpression of Bomanins specific to each challenge. Mechanistically, flies in which BomS6 is expressed in the nervous system exhibit an enhanced recovery from the tremors induced by injected verruculogen and display improved survival. Thus, innate immunity also protects the host against the action of microbial toxins through secreted peptides and thereby increases its resilience to infection.

Serial Quantitation of Plasma Microbial Cell-Free DNA Before and After Diagnosis of Pulmonary Invasive Mold Infections After Hematopoietic Cell Transplant.

BACKGROUND: Plasma microbial cell-free DNA sequencing (mcfDNA-Seq) is a noninvasive test for microbial diagnosis of invasive mold infection (IMI). The utility of mcfDNA-Seq for predicting IMI onset and the clinical implications of mcfDNA concentrations are unknown. METHODS: We retrospectively tested plasma from hematopoietic cell transplant (HCT) recipients with pulmonary IMI and ≥1 mold identified by mcfDNA-Seq in plasma collected within 14 days of clinical diagnosis. Samples collected from up to 4 weeks before and 4 weeks after IMI diagnosis were evaluated using mcfDNA-Seq. RESULTS: Thirty-five HCT recipients with 39 IMIs (16 Aspergillus and 23 non-Aspergillus infections) were included. Pathogenic molds were detected in 38%, 26%, 11%, and 0% of samples collected during the first, second, third, and fourth week before clinical diagnosis, respectively. In non-Aspergillus infections, median mcfDNA concentrations in samples collected within 3 days of clinical diagnosis were higher in infections with versus without extrapulmonary spread (4.3 vs 3.3 log10 molecules per microliter [mpm], P = .02), and all patients (8/8) with mcfDNA concentrations >4.0 log10 mpm died within 42 days after clinical diagnosis. CONCLUSIONS: Plasma mcfDNA-Seq can identify pathogenic molds up to 3 weeks before clinical diagnosis of pulmonary IMI. Plasma mcfDNA concentrations may correlate with extrapulmonary spread and mortality in non-Aspergillus IMI.

Fungal Melanin Rewires Macrophage Metabolism.

Aspergillus fumigatus is a deadly fungal pathogen in immunocompromised patients. A report by Gonçalves et al. reveals that melanin, a secondary metabolite present at the surface of infecting fungal spores, induces glycolysis in macrophages to promote inflammatory responses. This opens a window for the development of innovative host-directed antifungal therapies.

Breakthrough Invasive Fungal Infections in Patients With High-Risk Hematological Disorders Receiving Voriconazole and Posaconazole Prophylaxis: A Systematic Review.

BACKGROUND: Primary antifungal prophylaxis with mold-active azoles is used to prevent invasive fungal infections in patients with high-risk hematological disorders; however, breakthrough infections occur, and the reasons for treatment failure are still not fully understood. To help inform clinical decisions, we sought to define microbiological, clinical, and pharmacological characteristics of proven and probable breakthrough invasive fungal infections (bIFIs) in patients with high-risk hematological disorders receiving voriconazole or posaconazole prophylaxis. METHODS: We performed a systematic review of the literature following Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. The search strategy was last conducted on 19 April 2023. RESULTS: We assessed 5293 studies for eligibility, and 300 were selected for data extraction. These studies described 1076 cases of bIFIs occurring under voriconazole (42.5%) or posaconazole (57.5%). The most commonly found pathogens were Aspergillus (40%), Mucorales (20%), Candida (18%), and Fusarium (9%) species. Mucorales were more frequent among voriconazole-emerging cases, whereas Aspergillus and Fusarium were more prevalent among posaconazole-emerging cases. Definitive, putative, or probable antifungal resistance was found in 31% of cases. Therapeutic drug monitoring showed subtherapeutic azole concentration in 32 of 90 (36%) cases. Infection-related mortality was reported in 117 cases and reached 35%. CONCLUSIONS: In our systemic review, the most common bIFIs were aspergillosis, mucormycosis, candidiasis, and fusariosis. Antifungal resistance explains only a minority of cases. Subtherapeutic prophylaxis was frequent but rarely reported. Prospective studies are needed to better understand these infections and to establish optimal management.

Epidemiological and clinical features of a large blastomycosis outbreak at a paper mill in Michigan.

BACKGROUND: Blastomycosis is an environmentally acquired fungal infection that can result in severe pulmonary illness and high hospitalization rates. In 2023, a blastomycosis outbreak was detected among workers at a paper mill in Delta County, Michigan. METHODS: We included patients with clinical and laboratory evidence of blastomycosis who had spent ≥40 hours in Delta County since September 1, 2022 and had illness onset December 1, 2022-July 1, 2023. We assessed epidemiological and clinical features of patients and evaluated factors associated with hospitalization. We performed whole-genome sequencing to characterize genetic relatedness of clinical isolates from eight patients. RESULTS: In total, 131 patients were identified; all had worked at or visited the mill. Sixteen patients (12%) were hospitalized; one died. Compared with non-hospitalized patients, more hospitalized patients had diabetes (p=0.03) and urine antigen titers above the lower limit of quantification (p<0.001). Hospitalized patients were also more likely to have had ≥1 healthcare visits before receiving a blastomycosis diagnostic test (p=0.02) and to have been treated with antibiotics prior to antifungal prescription (p=0.001). All sequenced isolates were identified as Blastomyces gilchristii and clustered into a distinct outbreak cluster. CONCLUSIONS: This was the largest documented blastomycosis outbreak in the United States. Epidemiologic evidence indicated exposures occurred at or near the mill, and genomic findings suggested a common exposure source. Patients with diabetes may have increased risk for hospitalization, and elevated urine antigen titers could indicate greater disease severity. Early suspicion of blastomycosis may prompt earlier diagnosis and treatment, potentially reducing unnecessary antibiotic prescriptions and improving patient outcomes.

Incidence and risk factors for invasive fungal infections in patients initiating TNF-alpha inhibitors for inflammatory bowel disease and rheumatoid arthritis.

In a commercial claims database analysis, <0.5% of patients with inflammatory bowel disease or rheumatoid arthritis developed an IFI within one year of initiating TNF-alpha therapy. Histoplasmosis was the most common IFI type. Overall IFI incidence varied based on region, underlying conditions, and use of certain immunosuppressive medications.

Systematic Review of Prevalence of Histoplasma Antigenuria in Persons with HIV in Latin America and Africa.

Histoplasmosis is a fungal disease associated with substantial mortality rates among persons with advanced HIV disease. Our systematic review synthesized data on the global prevalence of Histoplasma--caused antigenuria in persons with HIV. We searched PubMed/Medline, Embase, and Scopus databases on January 3, 2023, to identify cross-sectional and cohort studies evaluating Histoplasma antigenuria prevalence among adults with HIV infection. We calculated point estimates and 95% CIs to summarize prevalence. Of 1,294 studies screened, we included 15. We found Histoplasma antigenuria among 581/5,096 (11%; 95% CI 11%-12%) persons with HIV and 483/3,789 persons with advanced HIV disease (13%; 95% CI 12%-14%). Among persons with HIV and symptoms consistent with histoplasmosis, Histoplasma antigenuria prevalence was 14% (95% CI 13%-15%; 502/3,631 participants). We determined that persons with advanced HIV disease, inpatients, and symptomatic persons might benefit from a systematic approach to early detection of histoplasmosis using urine antigen testing.

Rhizopus arrhizus infection in mice causes brain histopathological alterations and exacerbates neuronal apoptosis.

Rhizopus arrhizus is a fungus that can cause central nervous system infections in animals, resulting in high morbidity and mortality, but the mechanism of injury is rarely reported. In this study, we investigated the mechanism of Rhizopus arrhizus damage to the central nervous system of mice by observing the clinical neurological symptoms and resolving the pathological changes in the ultrastructure of brain tissues, combined with the alteration of apoptosis-related genes and immunohistochemistry (IHC). The results showed that all the mice in the treated group died, the brain pyknosis of neurons, there were black mycelium aggregates around the blood vessels, and apoptotic vesicles were produced. The RT-qPCR results showed that, compared with the control group, the relative transcriptome levels of Caspase 8 and BcL-2 genes were significantly increased (P < 0.05), the relative transcriptome level of Caspase 9 gene was highly significant (P < 0.01), the relative transcriptome level of Caspase 3 and Bax gene was significantly decreased (P < 0.05), and the ratio of Bcl-2/Bax was significantly increased (P < 0.05) in the brains of the treated group. TUNEL staining showed that the rate of neuronal apoptosis in the treated group of mice was extremely significantly higher than that in the control group (P < 0.01). This study shows that Rhizopus arrhizus strain XMLO1 causes brain damage by triggering neuronal apoptosis. This study provided a theoretical basis for revealing the mechanism of Rhizopus arrhizus infection.

Emerging roles of Dectin-1 in noninfectious settings and in the CNS.

Dectin-1 is a C-type lectin receptor (CLR) expressed on the surface of various mammalian myeloid cells. Dectin-1 recognizes ß-glucans and elicits antifungal proinflammatory immune responses. Recent studies have begun to examine the biology of Dectin-1 in previously less explored settings, such as homeostasis, sterile inflammation, and in the central nervous system. Indeed, in certain contexts, Dectin-1 is now known to promote tolerance, and anti-inflammatory and neuroprotective responses. In this review, we provide an overview of the current understanding of the roles of Dectin-1 in immunology beyond the context of fungal infections, mainly focusing on in vivo neuroimmunology studies, which could reveal new therapeutic approaches to modify innate immune responses in neurologic disorders.

Longitudinally extensive posterolateral myelitis revealing invasive CNS aspergillosis in an immunocompetent patient.

BACKGROUND: Fungal infections of the central nervous system usually affect immunocompromised patients. Primary Aspergillus myelitis has never been described. REPORT: A 45-year-old immunocompetent male with subacute paraplegia was treated for inflammatory myelitis before clinical deterioration requiring mechanical ventilation. Purulent meningitis preceded the formation of a paraspinal nodule biopsied by neurosurgery. Histopathological analysis revealed the presence of fungal hyphae, and polymerase chain reaction was positive for Aspergillus fumigatus. No cause of immunodeficiency was identified in this patient. DISCUSSION: Primary Aspergillus myelitis may be confused with inflammatory myelitis and should be considered even in the absence of apparent immunosuppression.

Surgical treatment of invasive fungal infection in pediatric oncology.

BACKGROUND: Invasive fungal disease (IFD) is typically aggressive and related with high mortality in children with a hematological malignancy. The association of medical and surgical treatment may ameliorate the outcome. The aim of this study was to analyze the surgical treatment of fungal infections in pediatric oncological populations. METHODS: Retrospective study (2000-2022) of a single-center experience. We reviewed the medical record of all patients with hematologic malignancies and IFD, analyzing the outcome. RESULTS: From the 70 pediatric cases of hematologic malignancies with the diagnosis IFD over 22 years, we included in the present study 44 cases who required surgical approaches for either diagnosis or treatment. Twenty-one patients were males and the mean age was 11 (range 1-23) years. The main indications for surgery were lack of improvement following medical treatment and/or progression of fungal infection (80%) and diagnosis confirmation (20%). Only five patients needed an emergency operation for rapid worsening of symptoms. The most common site of infection was the lung (80%) and the most frequently identified pathogen was Aspergillus (75%). The most common surgical procedures were lobectomy (performed in 17 patients) and atypical lung resection (10). Complications of surgery were mostly treated by medical approach. The mean time of resumption of oncological treatment was 40 (range 0-150) days. CONCLUSIONS: Surgery is an important step in the multimodal treatment of invasive fungal infection with excellent resolution rate. Overall mortality depends on the underlying malignancy.

Increase in the incidence of invasive fungal infections due to Volvariella Volvacea.

PURPOSE: We aimed to show the increasing incidence of invasive fungal infections due to Volvariella Volvacea in patients with immunosuppression. METHODS: We present a case of an invasive fungal infection caused by Volvariella volvacea, and summarize the clinical and pathological features based on this case and a review of the literature. RESULTS: A total of seven patients with IFIs due to Volvariella Volvacea have been reported in the literature. The majority of cases have been obtained between 2019 and 2022. Including our case, they all had acquired immunosuppression. The lung and brain were the most commonly affected organs. All eight of these patients received antifungal therapy, but five still died one to seven months after occurrences of IFIs. CONCLUSION: The incidence of invasive fungal infections due to Volvariella Volvacea is increasing in recent years. It mainly occurred in patients with immunosuppression, especially in patients with malignant hematological cancers, and increased mortality.

Candida auris: Understanding the dynamics of C. auris infection versus colonization.

Candida auris is a pathogen of growing public health concern worldwide. However, risk factors contributing to C. auris infection in patients colonized with C. auris remain unclear. Understanding these risk factors is crucial to prevent colonization-to-infection transition and devise effective preventive strategies. This study aimed to investigate risk factors associated with C. auris infection compared to colonization. The study included 97 patients who acquired laboratory-confirmed C. auris in either matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry or VITEK 2 system from October 2019 to June 2023. Baseline demographics and known risk factors associated with C. auris infection were collected from electronic medical records. The infection group had C. auris from a sterile site or non-sterile site with evidence of infection. The colonization group was followed up for a median of 30 days for any signs of infection. Associations between relevant variables and C. auris infection were assessed using multivariable logistic regression. The infection group (n = 31) was more likely to be bedbound, with longer hospital stays and more arterial catheters. Chronic kidney disease (odds ratio [OR] 45.070), carriage of multidrug-resistant organisms (OR 64.612), and vasopressor use for > 20 days (OR 68.994) were associated with C. auris infection, after adjusting for sex, age, and prior colonization with C. auris. Chronic kidney disease, carriage of multidrug-resistant organisms, and prolonged vasopressor use emerged as significant risk factors for C. auris infection compared to colonization. They could be used to predict C. auris infection early in patients colonized with C. auris.

Identifying risk factors for Candida auris infection should be an essential component of care in patients colonized with C. auris. Chronic kidney disease, carriage of multidrug-resistant organisms, and prolonged vasopressor use emerged as significant risk factors for C. auris infection.

Revealing the hidden interplay: The unexplored relationship between fungi and viruses beyond HIV, SARS-CoV-2, and influenza.

The complex interaction between viruses and fungi has profound implications, especially given the significant impact of these microorganisms on human health. While well-known examples such as HIV, influenza, and SARS-CoV-2 are recognized as risk factors for invasive fungal diseases, the relationship between viruses and fungi remains largely underexplored outside of these cases. Fungi and viruses can engage in symbiotic or synergistic interactions. Remarkably, some viruses, known as mycoviruses, can directly infect fungi, may influencing their phenotype and potentially their virulence. In addition, viruses and fungi can coexist within the human microbiome, a complex ecosystem of microorganisms. Under certain conditions, viral infection might predispose the host to an invasive fungal infection, as observed with influenza-associated pulmonary aspergillosis or COVID-19 associated pulmonary aspergillosis. We aim in this review to highlight potential connections between fungi and viruses (CMV and other herpesviruses, HTLV-1 and respiratory viruses), excluding SARS-CoV-2 and influenza.

The link between invasive fungal diseases and certain viruses (HIV, SARS-CoV-2 and influenza) is now well established. For other viruses, however, the relationship remains uncertain. In this review, we aim to highlight associations between fungi and viruses, except HIV, SARS-CoV-2 and influenza.

Mycology laboratory diagnostic capacity for invasive fungal diseases in public hospitals in Vietnam.

This was a cross-sectional study on the availability of laboratory infrastructure and capacity for the diagnosis of invasive fungal diseases in 24 public hospitals in Vietnam in 2023. Among the hospitals surveyed, 66.7% (14/21) had specialized personnel assigned for mycology testing, and 95.8% (23/24) had a separate microbiology laboratory space. Microscopy and culture methods are available in nearly all laboratories for isolate identification. Antifungal susceptibility testing is only performed for yeasts in 16/24 (66.7%) laboratories. Non-culture methods are hardly used in laboratories. Strengthening local laboratory capacities is essential to meeting health needs in these endemic regions.

There was a need for investment in fungal diagnostics to improve health services in the settings with a burden of endemic fungal infections.

Diagnostic performance of the (1-3)-ß-d-glucan assay in patients with different risks for invasive fungal diseases.

We evaluated the diagnostic performance of the ß-d-glucan (BDG) test (Beijing Gold Mountain River Tech) in diagnosing invasive fungal disease (IFD) and its variations among patients with different risks. Patients ≥18 years old who underwent a serum BDG test (positive cutoff value >80 pg/ml) from April 2017 through May 2018 were collected consecutively. Patients were classified into three groups: group 1, patients with host factors as defined by the prior 2008 European Organization for Research and Treatment (EORTC) criteria; group 2, those with extended host factors in 2020 EORTC criteria; and group 3, those without any risk factor mentioned in the criteria. IFD was defined by 2020 EORTC criteria, but BDG was not considered. Diagnostic performance of the serum BDG test was measured by the area under the curve (AUC) of the receiver-operating characteristic curve. Among 469 patients, 15.4% (72/469) were diagnosed with IFD (48/191 [25.1%], 14/144 [9.7%], and 10/134 [7.5%] in groups 1, 2, and 3, respectively). The BDG assay showed fair performance (AUC 0.748 [95% CI: 0.688-0.810]). Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were 77.8%, 60.7%, 26.4%, and 93.8%, respectively. PPV was higher in group 1, and NPV was higher in group 3. Additionally, diagnostic odds ratios were 6.73, 2.88, and 5.92 in groups 1, 2, and 3. Immunosuppressant use, non-IFD/Candida colonization, and central venous catheter were associated with false positivity. Clinicians should cautiously interpret the BDG assay, considering the various diagnostic performances depending on the different levels of risk.

We evaluated the diagnostic performance of the serum ß-d-glucan test in patients with varying risks for invasive fungal diseases. The test showed acceptable performance, but its predictive values differed among risk groups, highlighting the need for tailored interpretation.

Machine learning to identify clinically relevant Candida yeast species.

Fungal infections, especially due to Candida species, are on the rise. Multi-drug resistant organisms such as Candida auris are difficult and time consuming to identify accurately. Machine learning is increasingly being used in health care, especially in medical imaging. In this study, we evaluated the effectiveness of six convolutional neural networks (CNNs) to identify four clinically important Candida species. Wet-mounted images were captured using bright field live-cell microscopy followed by separating single-cells, budding-cells, and cell-group images which were then subjected to different machine learning algorithms (custom CNN, VGG16, ResNet50, InceptionV3, EfficientNetB0, and EfficientNetB7) to learn and predict Candida species. Among the six algorithms tested, the InceptionV3 model performed best in predicting Candida species from microscopy images. All models performed poorly on raw images obtained directly from the microscope. The performance of all models increased when trained on single and budding cell images. The InceptionV3 model identified budding cells of C. albicans, C. auris, C. glabrata (Nakaseomyces glabrata), and C. haemulonii in 97.0%, 74.0%, 68.0%, and 66.0% cases, respectively. For single cells of C. albicans, C. auris, C. glabrata, and C. haemulonii InceptionV3 identified 97.0%, 73.0%, 69.0%, and 73.0% cases, respectively. The sensitivity and specificity of InceptionV3 were 77.1% and 92.4%, respectively. Overall, this study provides proof of the concept that microscopy images from wet-mounted slides can be used to identify Candida yeast species using machine learning quickly and accurately.

Fungal infections due to Candida yeasts are increasing worldwide. Existing methods to identify these pathogens are difficult and time consuming. We find that machine learning can identify Candida species from images quickly and accurately, improving the diagnosis of infectious fungal diseases.