A Novel Deep Learning-Based Black Fungus Disease Identification Using Modified Hybrid Learning Methodology.

Currently, countries across the world are suffering from a prominent viral infection called COVID-19. Most countries are still facing several issues due to this disease, which has resulted in several fatalities. The first COVID-19 wave caused devastation across the world owing to its virulence and led to a massive loss in human lives, impacting the country's economy drastically. A dangerous disease called mucormycosis was discovered worldwide during the second COVID-19 wave, in 2021, which lasted from April to July. The mucormycosis disease is commonly known as "black fungus," which belongs to the fungus family Mucorales. It is usually a rare disease, but the level of destruction caused by the disease is vast and unpredictable. This disease mainly targets people already suffering from other diseases and consuming heavy medication to counter the disease they are suffering from. This is because of the reduction in antibodies in the affected people. Therefore, the patient's body does not have the ability to act against fungus-oriented infections. This black fungus is more commonly identified in patients with coronavirus disease in certain country. The condition frequently manifests on skin, but it can also harm organs such as eyes and brain. This study intends to design a modified neural network logic for an artificial intelligence (AI) strategy with learning principles, called a hybrid learning-based neural network classifier (HLNNC). The proposed method is based on well-known techniques such as convolutional neural network (CNN) and support vector machine (SVM). This article discusses a dataset containing several eye photographs of patients with and without black fungus infection. These images were collected from the real-time records of people afflicted with COVID followed by the black fungus. This proposed HLNNC scheme identifies the black fungus disease based on the following image processing procedures: image acquisition, preprocessing, feature extraction, and classification; these procedures were performed considering the dataset training and testing principles with proper performance analysis. The results of the procedure are provided in a graphical format with the precise specification, and the efficacy of the proposed method is established.

Resolving the equation between mucormycosis and COVID-19 disease.

The COVID-19 patients, both infected and recovered are rapidly contracting mucormycetes infections due to the 'Mucorales' order, under Zygomycetes class of fungi. The mucorales fungi commonly known to exist in our natural surroundings including soil, but the frequency of incidences was never rampant. This sudden spike in infections, is locally known as 'black fungus,' and is affecting various organs, including- eyes, sinuses, nose, brain, skin, intestine, lungs, etc. The severity of situation is ascertainable from the fact that, in certain cases surgical eye/jaws removal persists as the only viable option to avert mortality, as therapeutic interventions are limited. This epidemic situation intrigued experts to investigate the probable reason behind this unpredicted escalation in reported cases, including in recuperated COVID-19 patients, as person-to-person spread of infection is not common. The comparison of physiological parameters in healthy and COVID-19 afflicted patients highlights that the underlying conditions including diabetes mellitus, steroidal therapy, lymphopenia (decreased CD4+ and CD8+ lymphocytes), deregulated cytokine release storm, elevated free iron levels (hemosiderosis) in blood and insulin insensitivity are playing major roles in deteriorating conditions in rarely pathogenic fungal infections. This review is an attempt to explain the rationalities that makes people vulnerable to mucormycetes infection.

In Silico Designing of a Multitope Vaccine against Rhizopus microsporus with Potential Activity against Other Mucormycosis Causing Fungi.

During the current era of the COVID-19 pandemic, the dissemination of Mucorales has been reported globally, with elevated rates of infection in India, and because of the high rate of mortality and morbidity, designing an effective vaccine against mucormycosis is a major health priority, especially for immunocompromised patients. In the current study, we studied shared Mucorales proteins, which have been reported as virulence factors, and after analysis of several virulent proteins for their antigenicity and subcellular localization, we selected spore coat (CotH) and serine protease (SP) proteins as the targets of epitope mapping. The current study proposes a vaccine constructed based on top-ranking cytotoxic T lymphocyte (CTL), helper T lymphocyte (HTL), and B cell lymphocyte (BCL) epitopes from filtered proteins. In addition to the selected epitopes, ß-defensins adjuvant and PADRE peptide were included in the constructed vaccine to improve the stimulated immune response. Computational tools were used to estimate the physicochemical and immunological features of the proposed vaccine and validate its binding with TLR-2, where the output data of these assessments potentiate the probability of the constructed vaccine to stimulate a specific immune response against mucormycosis. Here, we demonstrate the approach of potential vaccine construction and assessment through computational tools, and to the best of our knowledge, this is the first study of a proposed vaccine against mucormycosis based on the immunoinformatics approach.

Human mucosal-associated invariant T cells respond to Mucorales species in a MR1-dependent manner.

Activation of mucosal-associated invariant T cells (MAIT cells) by certain bacteria, viruses, and yeast is well studied, but the activation potential of filamentous moulds from the order Mucorales is not known. Here, we show a rapid response of human MAIT cells against the Mucorales species Mucor circinelloides, Rhizopus arrhizus, and Rhizopus microsporus. This activation included upregulation of CD69 and degranulation marked by increased CD107a expression, while intracellular perforin and granzyme A expression were reduced. Furthermore, blocking of the antigen-presenting molecule major histocompatibility complex class I-related abrogated MAIT cell activation demonstrating a T cell receptor-dependent stimulation by Mucorales.

Invasive Mucormycosis in Children With Malignancies: Report From the Infection Working Group of the Hellenic Society of Pediatric Hematology-Oncology.

Mucormycosis is an invasive, life-threatening fungal infection that mainly affects immunocompromised hosts. We collected data of pediatric mucormycosis cases from all 7 Greek Hematology-Oncology Departments for the years 2008-2017. Six cases of invasive mucormycosis diagnosed during treatment for malignancies were included in the study. In 4 children (66%) mucormycosis occurred within the first 20 days after diagnosis of the underlying disease. Two cases were classified as proven mucormycosis and 4 as probable. The most frequently recorded species was Rhizopus arrhizus (2 patients), followed by Mucor spp (1), and Lichtheimia spp (1). All patients received liposomal amphotericin B. Combined antifungal treatment was used in 5 cases. Surgical excision was performed in 4 cases (66%). Two patients died at 6 and 12 months after the diagnosis, respectively, 1 (17%) because of mucormycosis. Our data suggest that mucormycosis may occur early after the initiation of intensive chemotherapy in children with malignancies.

Development and evaluation of a whole blood-based approach for flow cytometric quantification of CD154+ mould-reactive T cells.

CD154+ mould-reactive T cells were proposed as a novel biomarker in the diagnosis of invasive mycoses. As PBMC-based protocols for flow cytometric quantification of these cells are logistically challenging and susceptible to preanalytic delays, this study evaluated and optimized a whole blood-based method for the detection of mould-reactive T cells. Blood collection tubes containing costimulatory antibodies and Aspergillus fumigatus mycelial lysates were inoculated with heparinized whole blood from healthy adults, and detection rates of CD154+/CD4+A. fumigatus reactive T cells were compared with PBMC-based detection using samples from the same donors. In contrast to the PBMC-based method, double costimulation with αCD28 and αCD49d was crucial for reliable whole blood stimulation. Optimizing stimulation schemes for both matrixes, significantly higher specific T-cell detection rates were achieved by the whole blood-based method, whereas the unspecific background stimulation remained low. MHC II-dependent CD154+ upregulation was demonstrated for both matrixes. Excellent correlation and reproducible conversion factors between whole blood and PBMC-based results were observed. Using frozen ready-to-use test tubes containing costimulatory antibodies and lysates, detection rates of specific T cells were comparable to freshly prepared blood collection tubes. The optimized whole blood-based protocol was also used to detect Rhizopus arrhizus and Rhizomucor pusillus reactive T cells, resulting in 1.5- to 2.7-fold higher detection rates compared with PBMC-based measurement. In summary, the whole blood protocol is a robust, highly sensitive, and cost-effective method for mould-reactive T-cell quantification, allowing for point-of-care sample stimulation and contributing to better assay standardization in multi-centre evaluation of mould reactive T-cell quantification.

The geographical region of origin determines the phagocytic vulnerability of Lichtheimia strains.

Mucormycoses are life-threatening infections that affect patients suffering from immune deficiencies. We performed phagocytosis assays confronting various strains of Lichtheimia species with alveolar macrophages, which form the first line of defence of the innate immune system. To investigate 17 strains from four different continents in a comparative fashion, transmitted light and confocal fluorescence microscopy was applied in combination with automated image analysis. This interdisciplinary approach enabled the objective and quantitative processing of the big volume of image data. Applying machine-learning supported methods, a spontaneous clustering of the strains was revealed in the space of phagocytic measures. This clustering was not driven by measures of fungal morphology but rather by the geographical origin of the fungal strains. Our study illustrates the crucial contribution of machine-learning supported automated image analysis to the qualitative discovery and quantitative comparison of major factors affecting host-pathogen interactions. We found that the phagocytic vulnerability of Lichtheimia species depends on their geographical origin, where strains within each geographic region behaved similarly, but strongly differed amongst the regions. Based on this clustering, we were able to also classify clinical isolates with regard to their potential geographical origin.

Mould-reactive T cells for the diagnosis of invasive mould infection-A prospective study.

Invasive mould infections (IMI) in immunocompromised patients are difficult to diagnose. Early and targeted treatment is paramount, but minimally invasive tests reliably identifying pathogens are lacking. We previously showed that monitoring pathogen-specific CD4+T cells in peripheral blood using upregulation of induced CD154 positive lymphocytes can be used to diagnose acute IMI. Here, we validate our findings in an independent patient cohort. We stimulated peripheral blood cells from at-risk patients with Aspergillus spp. and Mucorales lysates and quantitated mould-reactive CD4/CD69/CD154 positive lymphocytes via flow cytometry. Mould-reactive lymphocytes were quantitated in 115 at-risk patients. In 38 (33%) patients, the test was not evaluable, mainly due to low T cell counts or non-reactive positive control. Test results were evaluable in 77 (67%) patients. Of these, four patients (5%) had proven IMI and elevated mould-reactive T cell signals. Of 73 (95%) patients without proven IMI, 59 (81%) had mould-reactive T cell signals within normal range. Fourteen (19%) patients without confirmed IMI showed elevated T cell signals and 11 of those received antifungal treatment. The mould-reactive lymphocyte assay identified presence of IMI with a sensitivity of 100% and specificity of 81%. The mould-reactive lymphocyte assay correctly identified all patients with proven IMI. Assay applicability is limited by low T cell counts during bone marrow suppression. The assay has the potential to support diagnosis of invasive mould infection to facilitate tailored treatment even when biopsies are contraindicated or cultures remain negative.

Pathogenicity patterns of mucormycosis: epidemiology, interaction with immune cells and virulence factors.

Fungi of the basal lineage order Mucorales are able to cause infections in animals and humans. Mucormycosis is a well-known, life-threatening disease especially in patients with a compromised immune system. The rate of mortality and morbidity caused by mucormycosis has increased rapidly during the last decades, especially in developing countries. The systematic, phylogenetic, and epidemiological distributions of mucoralean fungi are addressed in relation to infection in immunocompromised patients. The review highlights the current achievements in (i) diagnostics and management of mucormycosis, (ii) the study of the interaction of Mucorales with cells of the innate immune system, (iii) the assessment of the virulence of Mucorales in vertebrate and invertebrate infection models, and (iv) the determination of virulence factors that are key players in the infection process, for example, high-affinity iron permease (FTR1), spore coat protein (CotH), alkaline Rhizopus protease enzyme (ARP), ADP-ribosylation factor (ARF), dihydrolipoyl dehydrogenase, calcineurin (CaN), serine and aspartate proteases (SAPs). The present mini-review attempts to increase the awareness of these difficult-to-manage fungal infections and to encourage research in the detection of ligands and receptors as potential diagnostic parameters and drug targets.

Recent advances in the molecular diagnosis of mucormycosis.

INTRODUCTION: Fungal infection burden related to Mucorales has been on the rise with significant associated morbidity and mortality. The major obstacle in the management has been lack of a non-invasive rapid and a reliable diagnostic test. Developing a culture-independent biomarker for the early diagnosis of mucormycosis is a major unmet need in modern mycology. Several approaches have been developed, such as immunohistochemistry (IHC) that can confirm the histopathologic diagnosis of the invasive mold infection, polymerase chain reaction (PCR) on formalin-fixed paraffin-embedded (FFPE) or fresh tissue, body fluids such as bronchoalveolar fluid (BAL), and detection directly from serum/blood. Serologic tests, matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS), metabolomics and metagenomic shotgun sequencing are other evolving technologies. Area covered: In this review paper, we report the current status of the molecular diagnostics in the diagnosis of mucormycosis: serologic tests, IHC, PCR, protein-based with MALDI-TOF, metabolomics and metagenomic sequencing. Expert commentary: This review will conclude with an expert commentary on the potential uses/challenges of the currently available tests and the future of molecular diagnostics for mucormycosis.

Conservation of Mannan Synthesis in Fungi of the Zygomycota and Ascomycota Reveals a Broad Diagnostic Target.

Ascomycetes and zygomycetes account for the majority of (i) fungi responsible for cutaneous, subcutaneous, and invasive human fungal infections, (ii) plant fungal pathogens, (iii) fungi that threaten global biodiversity, (iv) fungal agents of agricultural spoilage, and (v) fungi in water-damaged buildings. Rapid recognition of fungal infection (or contamination) enables early treatment (or remediation). A bioinformatics search found homologues of Saccharomyces cerevisiae Mnn9p present in members of the Zygomycota and Ascomycota phyla and absent in members of the Chytridiomycota and Basidiomycota. Mnn9p is a component of the yeast mannan polymerization complex and is necessary for α-1,6 mannan production. A monoclonal antibody (2DA6) was produced that was reactive with purified mannans of Mucor, Rhizopus, Aspergillus, Fusarium, and Candida species. Experimentation using a 2DA6 antigen capture enzyme-linked immunosorbent assay (ELISA) and extracts of fungi from the four phyla found agreement between the presence or absence of Mnn9p homologues and production or lack of production of mannan reactive with 2DA6. Studies of cell extracts from yeast mannan mutants identified α-1,6 mannan as the epitope recognized by 2DA6. To translate this finding into a point-of-use diagnostic, a 2DA6 lateral flow immunoassay was constructed that detected mannan in (i) extracts of dermatophytes and fungi that produce trauma-related infection and (ii) tissue from plants infected with Grosmannia clavigera or Sclerotium cepivorum These studies (i) revealed that the conservation of α-1,6-linked mannan in fungi of the Zygomycota and Ascomycota can be exploited as a broad diagnostic target and (ii) have provided a means to detect that target in an immunoassay platform that is well suited for clinic or field use.IMPORTANCE A key question asked when faced with an infection, an infestation, or environmental damage is whether it is a fungus. Identification of fungi as the cause of the problem can lead to remediation or treatment. Zygomycetes and ascomycetes account for the vast majority of fungal causes of human, animal, and plant disease, large-scale biodiversity loss, agricultural spoilage, and contamination of water-damaged buildings. These studies revealed the conservation of a common cell wall structural component of zygomycetes and ascomycetes to be a diagnostic target applicable to multiple pathogenic fungi and have leveraged that insight for practical use. Monoclonal antibodies reactive with this pan-fungal structure were produced and used to construct immunoassays (including ELISA and lateral flow assay) for detection of a broad range of pathogenic fungi.

Mucorales spores induce a proinflammatory cytokine response in human mononuclear phagocytes and harbor no rodlet hydrophobins.

Mucormycoses are life-threatening infections in immunocompromised patients. This study characterizes the response of human mononuclear cells to different Mucorales and Ascomycota. PBMC, monocytes, and monocyte derived dendritic cells (moDCs) from healthy donors were stimulated with resting and germinated stages of Mucorales and Ascomycota. Cytokine response and expression of activation markers were studied. Both inactivated germ tubes and resting spores of Rhizopus arrhizus and other human pathogenic Mucorales species significantly stimulated mRNA synthesis and secretion of proinflammatory cytokines. Moreover, R. arrhizus spores induced the upregulation of co-stimulatory molecules on moDCs and a specific T-helper cell response. Removal of rodlet hydrophobins by hydrofluoric acid treatment of A. fumigatus conidia resulted in enhanced immunogenicity, whereas the cytokine response of PBMCs to dormant R. arrhizus spores was not influenced by hydrofluoric acid. Scanning electron micrographs of Mucorales spores did not exhibit any morphological correlates of rodlet hydrophobins. Taken together, this study revealed striking differences in the response of human mononuclear cells to resting stages of Ascomycota and Mucorales, which may be explained by absence of an immunoprotective hydrophobin layer in Mucorales spores.

Fungal peptides from pneumonitis hypersensitivity etiologic agents are able to induce specific cellular immune response.

PURPOSE: Hypersensitivity pneumonitis (HP) is an immunoallergic disease due to chronic exposure to high quantities of different microorganisms such as Mycobacterium immunogenum (Mi), a mycobacterium, and Lichtheimia corymbifera (Lc), a filamentous fungus. It has recently been demonstrated that the protein DLDH (dihydrolipoyl dehydrogenase), is common to these microorganisms. This study aimed to investigate the immune potential of overlapping peptide pools covering the MiDLDH and LcDLDH. EXPERIMENTAL DESIGN: A selection of 34 peptides, from the MiDLDH and LcDLDH, able to interact with Major Histocompatibility Complex (MHC) 1 and MHC 2, was obtained using three different epitope prediction websites. By means of ELISPOT assays, we compared the frequency of Interferon gamma (IFNγ) secreting peripheral blood mononuclear cells (PBMC) after stimulation with overlapping peptide pools. Tests were performed using cells from 35 healthy blood donors. RESULTS: One peptide pool containing five peptides from MiDLDH and able to interact with MHC 2 induced a marked IFNγ specific immune response (Pool F, p<0.001, Wilcoxon signed-rank test). CONCLUSION: This study demonstrated that peptides from microorganisms involved in HP were able to induce a high IFNγ specific immune response after stimulation of PBMCs from healthy blood donors which could be useful to develop an effective prevention strategy.

Filamentous Fungi.

Filamentous mycoses are often associated with significant morbidity and mortality. Prompt diagnosis and aggressive treatment are essential for good clinical outcomes in immunocompromised patients. The host immune response plays an essential role in determining the course of exposure to potential fungal pathogens. Depending on the effectiveness of immune response and the burden of organism exposure, fungi can either be cleared or infection can occur and progress to a potentially fatal invasive disease. Nonspecific cellular immunity (i.e., neutrophils, natural killer [NK] cells, and macrophages) combined with T-cell responses are the main immunologic mechanisms of protection. The most common potential mold pathogens include certain hyaline hyphomycetes, endemic fungi, the Mucorales, and some dematiaceous fungi. Laboratory diagnostics aimed at detecting and differentiating these organisms are crucial to helping clinicians make informed decisions about treatment. The purpose of this chapter is to provide an overview of the medically important fungal pathogens, as well as to discuss the patient characteristics, antifungal-therapy considerations, and laboratory tests used in current clinical practice for the immunocompromised host.

Mucorales-Specific T Cells in Patients with Hematologic Malignancies.

BACKGROUND: Invasive mucormycosis (IM) is an emerging life-threatening fungal infection. It is difficult to obtain a definite diagnosis and to initiate timely intervention. Mucorales-specific T cells occur during the course of IM and are involved in the clearance of the infection. We have evaluated the feasibility of detecting Mucorales-specific T cells in hematological patients at risk for IM, and have correlated the detection of such cells with the clinical conditions of the patients. METHODS AND FINDINGS: By using an enzyme linked immunospot assay, the presence of Mucorales-specific T cells in peripheral blood (PB) samples has been investigated at three time points during high-dose chemotherapy for hematologic malignancies. Mucorales-specific T cells producing interferon-γ, interleukin-10 and interleukin-4 were analysed in order to detect a correlation between the immune response and the clinical picture. Twenty-one (10.3%) of 204 patients, accounting for 32 (5.3%) of 598 PB samples, tested positive for Mucorales-specific T cells. Two groups could be identified. Group 1, including 15 patients without signs or symptoms of invasive fungal diseases (IFD), showed a predominance of Mucorales-specific T cells producing interferon-gamma. Group 2 included 6 patients with a clinical picture consistent with invasive fungal disease (IFD): 2 cases of proven IM and 4 cases of possible IFD. The proven patients had significantly higher number of Mucorales-specific T cells producing interleukin-10 and interleukin-4 and higher rates of positive samples by using derived diagnostic cut-offs when compared with the 15 patients without IFD. CONCLUSIONS: Mucorales-specific T cells can be detected and monitored in patients with hematologic malignancies at risk for IM. Mucorales-specific T cells polarized to the production of T helper type 2 cytokines are associated with proven IM and may be evaluated as a surrogate diagnostic marker for IM.