Human Fungal Pathogen Research Articles

Pleiotropic roles of LAMMER kinase, Lkh1 in stress responses and virulence of Cryptococcus neoformans

Dual-specificity LAMMER kinases are highly evolutionarily conserved in eukaryotes and play pivotal roles in diverse physiological processes, such as growth, differentiation, and stress responses. Although the functions of LAMMER kinase in fungal pathogens in pathogenicity and stress responses have been characterized, its role in Cryptococcus neoformans, a human fungal pathogen and a model yeast of basidiomycetes, remains elusive. In this study, we identified a LKH1 homologous gene and constructed a strain with a deleted LKH1 and a complemented strain. Similar to other fungi, the lkh1Δ mutant showed intrinsic growth defects. We observed that C. neoformans Lkh1 was involved in diverse stress responses, including oxidative stress and cell wall stress. Particularly, Lkh1 regulates DNA damage responses in Rad53-dependent and -independent manners. Furthermore, the absence of LKH1 reduced basidiospore formation. Our observations indicate that Lkh1 becomes hyperphosphorylated upon treatment with rapamycin, a TOR protein inhibitor. Notably, LKH1 deletion led to defects in melanin synthesis and capsule formation. Furthermore, we found that the deletion of LKH1 led to the avirulence of C. neoformans in a systemic cryptococcosis murine model. Taken together, Lkh1 is required for the stress response, sexual differentiation, and virulence of C. neoformans.

The effect of infection with the entomopathogenic fungus Conidiobolus coronatus (Entomopthorales) on eighteen cytokine-like proteins in Galleria mellonella (Lepidoptera) larvae

BackgroundIn response to the replace mammal research models with insects in preliminary immunological studies, interest has grown in invertebrate defense systems. The immunological response is regulated by cytokines; however, while their role in mammals is well understood, little is known of their function in insects. A suitable target for studies into insect immunology is Galleria mellonella (Lepidoptera), the wax moth: a common host for human fungal and bacterial pathogens. G. mellonella is also a perfect subject for studies into the presence of cytokine-like proteins.Specific objectivesThe main goal of present research was detection in insect immunocompetent cells the 18 mammalian cytokines (IL-1α, IL-1β, IL-2, IL-3, IL-6, IL-7, IL-8, IL-12, IL-13, IL-15, IL-17, IL-19, IFN-γ, TNF-α, TNF-β, GM-CSF, M-CSF, G-CSF), which play important role in immunological response and indication how their level change after fungal infection.MethodologyThe changes of cytokine-like proteins level were detected in hemocytes taken from G. mellonella larvae infected with entomopathogenic fungus, C. coronatus. The presence of cytokine-proteins was confirmed with using fluorescence microscopy (in cultured hemocytes) and flow cytometry (in freshly collected hemolymph). The ELISA test was used to detect changes in concentration of examined cytokine-like proteins.ResultsOur findings indicated the presence of eighteen cytokine-like molecules in G. mellonella hemocytes during infection with C. coronatus. The hemocytes taken from infected larvae demonstrated higher fluorescence intensity for six cytokine-like proteins (GM-CSF, M-CSF, IL-3, IL-15, IL-1β and IL-19) compared to untreated controls. ELISA test indicated significantly higher IL-3 and IL-15. M-CSF, IL-1α and IL-19 concentration in the hemolymph after fungal infection, and significantly lower TNF-β and G-CSF.ConclusionsOur findings confirm that the selected cytokine-like molecules are present in insect hemocytes and that their concentrations change after fungal infection, which might suggest that they play a role in the anti-fungal immunological response.

The StuA Transcription Factor and Alternative Splicing Mechanisms Drive the Levels of MAPK Hog1 Transcripts in the Dermatophyte Trichophyton rubrum.

Trichophyton rubrum is a human fungal pathogen that causes dermatophytosis, an infection that affects keratinized tissues. Integrated molecular signals coordinate mechanisms that control pathogenicity. Transcriptional regulation is a core regulation of relevant fungal processes. Previous RNA sequencing data revealed that the absence of the transcription factor StuA resulted in the differential expression of the MAPK-related high glycerol osmolarity gene (hog1) in T. rubrum. Here we validated the role of StuA in regulating the transcript levels of hog1. We showed through RT-qPCR that transcriptional regulation controls hog1 levels in response to glucose, keratin, and co-culture with human keratinocytes. In addition, we also detected hog1 pre-mRNA transcripts that underwent alternative splicing, presenting intron retention in a StuA-dependent mechanism. Our findings suggest that StuA and alternative splicing simultaneously, but not dependently, coordinate hog1 transcript levels in T. rubrum. As a means of preventing and treating dermatophytosis, our results contribute to the search for new potential drug therapies based on the molecular aspects of signaling pathways in T. rubrum.

Synthesis, in-vitro and molecular docking studies of 3,4- dihydropyrmidine-2-(1H) one-based Calix[4]arenes as antimicrobial agent and kinase inhibitor

Calix[n]arenes, nowadays, are used as a fully developed synthetic scaffold that provides a variety of spatial arrangement in structural groups which makes them more sufficient to use as a bed to be linked to other important derivatives like dihydropyrimidinone compounds. Recently, great interest has been attracted towards in the development and recognition of dihydropyrimidine derivatives based on calixarene compounds. In this study, A series of derivatives consisting of tetra-tert-butylcalix[4]arene attached to 3,4-dihydropyrimidine-2-(1H)-one at the lower rim were successfully synthesized. This was achieved through the reaction between 6-bromomethyl-DHPMs compounds (5a-d) and tetra-tert-butyl calix[4]arene. The synthesized compounds showed antibacterial and antifungal activities. Compound e exhibited the strongest antibacterial effects among these compounds. The results regarding the antifungal activity suggest that the synthesized compounds, particularly compound c, have significant effectiveness in eradicating fungal pathogens in humans and showed promising performance in this study. Additionally, a molecular docking study of these compounds was conducted, revealing that the novel derivatives d and e act as highly potent inhibitors of EGFR mutants with a unique chemical structure.The synthesized compounds were subjected to characterization using various techniques, including FT-IR spectroscopy, 1H NMR spectroscopy, 13C NMR spectroscopy, and elemental analysis. These analyses provided spectral data and elemental composition information, allowing for a comprehensive characterization of the synthesized compounds (7a-d).

Isotope labeled 3D-Raman confocal imaging and atomic force microscopy study on epithelial cells interacting with the fungus Candida albicans

The human pathogenic fungus Candida albicans damages epithelial cells during superficial infections. Here we use three-dimensional-sequential-confocal Raman spectroscopic imaging and atomic force microscopy to investigate the interaction of C. albicans wild type cells, the secreted C. albicans peptide toxin candidalysin and mutant cells lacking candidalysin with epithelial cells. The candidalysin responsible for epithelial cell damage and exhibits in its deuterated form an identifiable Raman signal in a frequency region distinct from the cellular frequency region. Vibration modes at 2100–2200 cm−1 attributed to carbon‑deuterium bending and at 477 cm−1, attributed to the nitrogen‑deuterium out-of-plane bending, found around the nucleus, can be assigned to deuterated candidalysin. Atomic force microscopy visualized 100 nm deep lesions on the cell and force-distance curves indicate the higher adhesion on pore surrounding after incubation with candidalysin. Candidalysin targets the plasma membrane, but is also found inside of the cytosol of epithelial cells during C. albicans infection.

The EH domain-containing protein, EdeA, is involved in endocytosis, cell wall integrity, and pathogenicity in Aspergillus fumigatus.

Aspergillus fumigatus is a significant human pathogenic fungus known to cause invasive aspergillosis, a disease with a high mortality rate. Understanding the basic principles of A. fumigatus pathogenicity is crucial for developing effective strategies against this pathogen. Previous research has underscored the importance of endocytosis in the infection capacity of pathogenic yeasts; however, its biological function in pathogenic mold remains largely unexplored. Our characterization of EdeA in A. fumigatus sheds light on the role of endocytosis in the development, stress response, and pathogenicity of pathogenic molds. These findings suggest that the components of the endocytosis process may serve as potential targets for antifungal therapy.

The Putative Cytochrome b5 Domain-Containing Protein CaDap1 Homologue Is Involved in Antifungal Drug Tolerance, Cell Wall Chitin Maintenance, and Virulence in Candida albicans

Candida albicans (Ca), a prominent opportunistic fungal pathogen in humans, has garnered considerable attention due to its infectious properties. Herein, we have identified and characterized CaCDAP1 (Ca orf19.1034), a homolog of ScDAP1 found in Saccharomyces cerevisiae. CaCDAP1 encodes a 183-amino acid protein with a conserved cytochrome b5-like heme-binding domain. The deletion of CaDAP1 renders Ca cells susceptible to caspofungin and terbinafine. CaDAP1 deletion confers resistance to Congo Red and Calcofluor White, and sensitivity to sodium dodecyl sulfate. The deletion of CaDAP1 results in a 50% reduction in chitin content within the cell wall, the downregulation of phosphorylation levels in CaMkc1, and the upregulation of phosphorylation levels in CaCek1. Notably, CaDAP1 deletion results in the abnormal hyphal development of Ca cells and diminishes virulence in a mouse systemic infection model. Thus, CaDAP1 emerges as a critical regulator governing cellular responses to antifungal drugs, the synthesis of cell wall chitin, and virulence in Ca.

The narrow footprint of ancient balancing selection revealed by heterokaryon incompatibility genes in Aspergillus fumigatus.

In fungi, fusion between individuals leads to localized cell death, a phenomenon termed heterokaryon incompatibility. Generally, the genes responsible for this incompatibility are observed to be under balancing selection resulting from negative frequency-dependent selection. Here, we assess this phenomenon in Aspergillus fumigatus, a human pathogenic fungus with a very low level of linkage disequilibrium as well as an extremely high crossover rate. Using complementation of auxotrophic mutations as an assay for hyphal compatibility, we screened sexual progeny for compatibility to identify genes involved in this process, called het genes. In total, 5/148 (3.4%) offspring were compatible with a parent and 166/2142 (7.7%) sibling pairs were compatible, consistent with several segregating incompatibility loci. Genetic mapping identified five loci, four of which could be fine mapped to individual genes, of which we tested three through heterologous expression, confirming their causal relationship. Consistent with long-term balancing selection, trans-species polymorphisms were apparent across several sister species, as well as equal allele frequencies within A. fumigatus. Surprisingly, a sliding window genome-wide population-level analysis of an independent dataset did not show increased Tajima's D near these loci, in contrast to what is often found surrounding loci under balancing selection. Using available de novo assemblies, we show that these balanced polymorphisms are restricted to several hundred base pairs flanking the coding sequence. In addition to identifying the first het genes in an Aspergillus species, this work highlights the interaction of long-term balancing selection with rapid linkage disequilibrium decay.

Azole resistance mechanisms and population structure of the human pathogen Aspergillus fumigatus on retail plant products.

Aspergillus fumigatus is a ubiquitous saprotroph and human-pathogenic fungus that is life-threatening to the immunocompromised. Triazole-resistant A. fumigatus was found in patients without prior treatment with azoles, leading researchers to conclude that resistance had developed in agricultural environments where azoles are used against plant pathogens. Previous studies have documented azole-resistant A. fumigatus across agricultural environments, but few have looked at retail plant products. Our objectives were to determine if azole-resistant A. fumigatus is prevalent in retail plant products produced in the United States (U.S.), as well as to identify the resistance mechanism(s) and population genetic structure of these isolates. Five hundred twenty-five isolates were collected from retail plant products and screened for azole resistance. Twenty-four isolates collected from compost, soil, flower bulbs, and raw peanuts were pan-azole resistant. These isolates had the TR34/L98H, TR46/Y121F/T289A, G448S, and H147Y cyp51A alleles, all known to underly pan-azole resistance, as well as WT alleles, suggesting that non-cyp51A mechanisms contribute to pan-azole resistance in these isolates. Minimum spanning networks showed two lineages containing isolates with TR alleles or the F46Y/M172V/E427K allele, and discriminant analysis of principle components identified three primary clusters. This is consistent with previous studies detecting three clades of A. fumigatus and identifying pan-azole-resistant isolates with TR alleles in a single clade. We found pan-azole resistance in U.S. retail plant products, particularly compost and flower bulbs, which indicates a risk of exposure to these products for susceptible populations and that highly resistant isolates are likely distributed worldwide on these products.IMPORTANCEAspergillus fumigatus has recently been designated as a critical fungal pathogen by the World Health Organization. It is most deadly to people with compromised immune systems, and with the emergence of antifungal resistance to multiple azole drugs, this disease carries a nearly 100% fatality rate without treatment or if isolates are resistant to the drugs used to treat the disease. It is important to determine the relatedness and origins of resistant A. fumigatus isolates in the environment, including plant-based retail products, so that factors promoting the development and propagation of resistant isolates can be identified.

A Lateral-Flow Device for the Rapid Detection of Scedosporium Species.

Scedosporium species are human pathogenic fungi, responsible for chronic, localised, and life-threatening disseminated infections in both immunocompetent and immunocompromised individuals. The diagnosis of Scedosporium infections currently relies on non-specific CT, lengthy and insensitive culture from invasive biopsy, and the time-consuming histopathology of tissue samples. At present, there are no rapid antigen tests that detect Scedosporium-specific biomarkers. Here, we report the development of a rapid (30 min) and sensitive (pmol/L sensitivity) lateral-flow device (LFD) test, incorporating a Scedosporium-specific IgG1 monoclonal antibody (mAb), HG12, which binds to extracellular polysaccharide (EPS) antigens between ~15 kDa and 250 kDa secreted during the hyphal growth of the pathogens. The test is compatible with human serum and allows for the detection of the Scedosporium species most frequently reported as agents of human disease (Scedosporium apiospermum, Scedosporium aurantiacum, and Scedosporium boydii), with limits of detection (LODs) of the EPS biomarkers in human serum of ~0.81 ng/mL (S. apiospermum), ~0.94 ng/mL (S. aurantiacum), and ~1.95 ng/mL (S. boydii). The Scedosporium-specific LFD (ScedLFD) test therefore provides a potential novel opportunity for the detection of infections caused by different Scedosporium species.

Thermotolerance of environmental fungi captured from indoor air.

While more than six million species make up the fungal kingdom, less than 300 cause disease in humans, in part because many fungal species are unable to grow at temperatures at or above human body temperature. However, there is concern that as global temperatures increase, fungal species that do not currently pose a risk to human health may adapt to growth at higher temperatures, opening the door for the emergence of new human fungal pathogens. This study assessed the growth of five fungal isolates captured from indoor air at temperatures up to 38°C.  All five isolates grew well at 24-27°C, grew poorly or not at all at 36°C, and none grew at 38°C.  We then investigated the growth of three fungal isolates captured from indoor air at 36 - 39°C when the growth temperature was gradually increased from ambient room temperature to 36 - 39°C, compared to growth after a sudden change of temperature from room temperature to 36 - 39°C.  All three isolates still exhibit poor to no growth at 39°C.  This research was supported by a NASA-WVSGC Undergraduate Research Fellowship (AEC) and a NASA-WVSGC Research Enhancement Award (LSR).

Identification of an active RNAi pathway in Candida albicans

RNA interference (RNAi) is a fundamental regulatory pathway with a wide range of functions, including regulation of gene expression and maintenance of genome stability. Although RNAi is widespread in the fungal kingdom, well-known species, such as the model yeast Saccharomyces cerevisiae, have lost the RNAi pathway. Until now evidence has been lacking for a fully functional RNAi pathway in Candida albicans, a human fungal pathogen considered critically important by the World Health Organization. Here, we demonstrated that the widely used C. albicans reference strain (SC5314) contains an inactivating missense mutation in the gene encoding for the central RNAi component Argonaute. In contrast, most other C. albicans isolates contain a canonical Argonaute protein predicted to be functional and RNAi-active. Indeed, using high-throughput small and long RNA sequencing combined with seamless CRISPR/Cas9-based gene editing, we demonstrate that an active C. albicans RNAi machinery represses expression of subtelomeric gene families. Thus, an intact and functional RNAi pathway exists in C. albicans, highlighting the importance of using multiple reference strains when studying this dangerous pathogen.

Phytochemical Composition, Antioxidant and Antifungal Activity of Essential Oils and Crude Extracts of <em>Dittrichia viscosa maritime</em> (L.), an Aromatic and Medicinal Plant from Northern Morocco

Dittrichia viscosa, a perennial herb belonging to the Asteraceae family, was collected in a coastal site located in northern Morocco. This study focused on exploring the phytochemical composition and biological activities of essential oils (EOs) and solvents (methanol and acetone) extract of leaves, stems, and flowers. Total phenolic (TPC) and total flavonoid (TFC) contents were determined, and the result showed a high level of TPC and TFC in leaves with the values of 127.61 mg GAEs/g and 117.82 mg CEs/g, respectively. HPLC-MS analysis highlighted 18 phenolic acids and flavonoids, with notable quantities of apigenin 7-glucoside (2233 μg/g) and chlorogenic acid (1429 μg/g). GC‒MS analysis of EOs allowed the identification of 27 biologically active compounds, dominated by decanoic acid (20.98%) and toluene (19.66%). Regarding the biological activities, antioxidant activity was evaluated through the DPPH test, while antifungal activity was tested using the microdilution method. The most potent antioxidant effect was observed in the methanol extract of leaves, with a value of 0.18 mg/mL, while the weakest effect was noted in the acetone extract of flowers, registering a value of 1.63 mg/mL. Similarly, the antioxidant activity of methanolic extracts surpassed that of acetonic extracts in each part of D. viscosa. Concerning antifungal activity, the findings reveal that all extracts demonstrated important capacity against the tested pathogens. The methanolic extract displayed high efficacy compared to the acetonic one, with MFC values ranging between 1.94-3.38 mg/mL and 2.88-4.75 for Candida albicans, Aspergillus niger and Trichophyton rubrum, respectively. However, for Fusarium oxysporum f. sp. albedinis, the MFCs were higher (7.5-8.5 mg/mL). Regarding EOs, the MFCs were more favorable for the three human pathogenic fungi, ranging between 1.88 and 3.35 µL/mL, compared to F. oxysporum, which has an MFCs of 3.5 µL/mL. In summary, the findings demonstrated that D. viscosa could be considered a useful alternative and reliable source of bioactive compounds for the pharmaceutical and agricultural industries.

A highly conserved tRNA modification contributes to C. albicans filamentation and virulence.

tRNA modifications play important roles in maintaining translation accuracy in all domains of life. Disruptions in the tRNA modification machinery, especially of the anticodon stem loop, can be lethal for many bacteria and lead to a broad range of phenotypes in baker's yeast. Very little is known about the function of tRNA modifications in host-pathogen interactions, where rapidly changing environments and stresses require fast adaptations. We found that two closely related fungal pathogens of humans, the highly pathogenic Candida albicans and its much less pathogenic sister species, Candida dubliniensis, differ in the function of a tRNA-modifying enzyme. This enzyme, Hma1, exhibits species-specific effects on the ability of the two fungi to grow in the hypha morphology, which is central to their virulence potential. We show that Hma1 has tRNA-threonylcarbamoyladenosine dehydratase activity, and its deletion alters ribosome occupancy, especially at 37°C-the body temperature of the human host. A C. albicans HMA1 deletion mutant also shows defects in adhesion to and invasion into human epithelial cells and shows reduced virulence in a fungal infection model. This links tRNA modifications to host-induced filamentation and virulence of one of the most important fungal pathogens of humans.IMPORTANCEFungal infections are on the rise worldwide, and their global burden on human life and health is frequently underestimated. Among them, the human commensal and opportunistic pathogen, Candida albicans, is one of the major causative agents of severe infections. Its virulence is closely linked to its ability to change morphologies from yeasts to hyphae. Here, this ability is linked-to our knowledge for the first time-to modifications of tRNA and translational efficiency. One tRNA-modifying enzyme, Hma1, plays a specific role in C. albicans and its ability to invade the host. This adds a so-far unknown layer of regulation to the fungal virulence program and offers new potential therapeutic targets to fight fungal infections.

A dual-targeting antifungal is effective against multidrug-resistant human fungal pathogens.

Drug-resistant fungal infections pose a significant threat to human health. Dual-targeting compounds, which have multiple targets on a single pathogen, offer an effective approach to combat drug-resistant pathogens, although ensuring potent activity and high selectivity remains a challenge. Here we propose a dual-targeting strategy for designing antifungal compounds. We incorporate DNA-binding naphthalene groups as the hydrophobic moieties into the host defence peptide-mimicking poly(2-oxazoline)s. This resulted in a compound, (Gly0.8Nap0.2)20, which targets both the fungal membrane and DNA. This compound kills clinical strains of multidrug-resistant fungi including Candida spp., Cryptococcus neoformans, Cryptococcus gattii and Aspergillus fumigatus. (Gly0.8Nap0.2)20 shows superior performance compared with amphotericin B by showing not only potent antifungal activities but also high antifungal selectivity. The compound also does not induce antimicrobial resistance. Moreover, (Gly0.8Nap0.2)20 exhibits promising in vivo therapeutic activities against drug-resistant Candida albicans in mouse models of skin abrasion, corneal infection and systemic infection. This study shows that dual-targeting antifungal compounds may be effective in combating drug-resistant fungal pathogens and mitigating fungal resistance.

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.

Pan-azole- and multi-fungicide-resistant Aspergillus fumigatus is widespread in the United States.

Aspergillus fumigatus is an important global fungal pathogen of humans. Azole drugs are among the most effective treatments for A. fumigatus infection. Azoles are also widely used in agriculture as fungicides against fungal pathogens of crops. Azole-resistant A. fumigatus has been increasing in Europe and Asia for two decades where clinical resistance is thought to be driven by agricultural use of azole fungicides. The most prevalent mechanisms of azole resistance in A. fumigatus are tandem repeats (TR) in the cyp51A promoter coupled with mutations in the coding region which result in resistance to multiple azole drugs (pan-azole resistance). Azole-resistant A. fumigatus has been isolated from patients in the United States (U.S.), but little is known about its environmental distribution. To better understand the distribution of azole-resistant A. fumigatus in the U.S., we collected isolates from agricultural sites in eight states and tested 202 isolates for sensitivity to azoles. We found azole-resistant A. fumigatus in agricultural environments in seven states showing that it is widespread in the U.S. We sequenced environmental isolates representing the range of U.S. sample sites and compared them with publicly available environmental worldwide isolates in phylogenetic, principal component, and ADMIXTURE analyses. We found worldwide isolates fell into three clades, and TR-based pan-azole resistance was largely in a single clade that was strongly associated with resistance to multiple agricultural fungicides. We also found high levels of gene flow indicating recombination between clades highlighting the potential for azole-resistance to continue spreading in the U.S.IMPORTANCEAspergillus fumigatus is a fungal pathogen of humans that causes over 250,000 invasive infections each year. It is found in soils, plant debris, and compost. Azoles are the first line of defense antifungal drugs against A. fumigatus. Azoles are also used as agricultural fungicides to combat other fungi that attack plants. Azole-resistant A. fumigatus has been a problem in Europe and Asia for 20 years and has recently been reported in patients in the United States (U.S.). Until this study, we did not know much about azole-resistant A. fumigatus in agricultural settings in the U.S. In this study, we isolated azole-resistant A. fumigatus from multiple states and compared it to isolates from around the world. We show that A. fumigatus which is resistant to azoles and to other strictly agricultural fungicides is widespread in the U.S.

Emerging and reemerging human fungal pathogens that affect people with weakened immune systems: a systematic review

BACKGROUNDEmerging and reemerging human fungal pathogens are becoming more closely associated with morbidity and mortality, with 13 million infections and 1.5 million deaths per year. They are most often associated with critically ill and immunosuppressed patients. Therefore, this systematic review focused on emerging and reemerging human fungal pathogens that affect immunosuppressed individuals. METHODS A systematic literature search was performed using PubMed, ScienceDirect, Web of Science, Google Scholar, and other sources (Google engine and manual search using a reference list). The data were extracted in a structured format prepared using Microsoft Excel. RESULTSCryptococcus neoformans, Candida auris, Aspergillus fumigatus, Candida albicans, Nakaseomyces glabrata (Candida glabrata), Histoplasma spp., Mucorales (Rhizopus spp., Mucor spp., Lichtheimia spp., and others), Fusarium spp., Candida tropicalis, Candida parapsilosis, Pichia kudriavzevii (Candida krusei), Talaromyces marneffei and Pneumocystis jirovecii were emerging and reemerging fungal pathogens reported among critically ill and immunocompromised patients including but not limited to HIV patients and patients with infectious diseases such as influenza, COVID-19, and tuberculosis as well as chronic conditions or co-morbidities such as asthma, hepatic cirrhosis, cancer, diabetes, cystic fibrosis (CF), transplant recipients, and chronic obstructive pulmonary disease (COPD). Climate change, agricultural activities, occupational hazards, deforestation, migratory trends of people, soil dispersion, decreased immunity of patients, biofilm development, medication tolerance, and resistance to antifungal therapies are all factors that contribute to the emergence of fungal diseases. CONCLUSIONThis review makes recommendations for policymakers, public health experts, and other stakeholders to improve the response to these fungal infections, including laboratory capacity and surveillance, fostering sustainable research and innovation, implementing public health initiatives, and limiting the development of antifungal drug resistance.

Cryptococcal meningitis presenting as simple headache in early adulthood HIV-infected patient: A case report

Cryptococcal meningitis is a rare disease caused by Cryptococcus neoformans or Cryptococcus gatii as the fungal pathogen in humans. The disease is highly prevalence in people living with human immunodeficiency virus (HIV) and is the major cause of mortality in an immunocompromised individual. This life-threatening disease does not often occur with specific signs and symptoms. We reported a case of an early adulthood HIV-infected patient with a mild headache and low-grade fever. There were no altered mental status or other significant specific manifestations. Blood examination, chest radiograph, and computed tomography scan revealed no significant results. C. neoformans were isolated from a culture of the cerebrospinal fluid specimen as the conventional gold standard examination for cryptococcal meningitis. A positive result was confirmed in the India ink staining preparation. The patient was treated with fluconazole monotherapy for the induction and consolidation phase and showed a positive disease progression during observations.

Antifungal Activity of Trichoderma orientale FMR 12486 Crude Extract against Some Human Pathogenic Fungi

     This work aimed to investigate the prevalence of pathogenic fungi and evaluate the antifungal activity of Trichoderma orientale FMR12486 crude extract against pathogenic fungi isolated from patients attending the National Center for Thoracic and Respiratory Diseases (having a history of tuberculosis) and consultant of Dermatology of Baghdad hospital, Iraq. A total of 80 clinical specimens were collected: 20 skin scrapings specimens and 60 sputum specimens. The results of direct examination by KOH 10% and culture showed that 11 (55%) cases from 20 skin specimens were positive for fungal infections, while in the sputum specimens, 28 (47%) cases from 60 were positive. Candida albicans represented the most common fungal infection isolated from sputum specimens which represented 18 cases at a rate of 64.2%, followed by Candida tropicalis 5 (17.9%), Aspergillus fumigates 2 (7.1%), Aspergillus flavus 1 (3.6%), Candida glabrata 1 (3.6%) and Candida krusei 1 (3.6%). While in the skin scrapes, Candida parapsilosis was the common which represented 3 cases at a rate of 27.3%, followed by Trichophyton mentagrophyte 2 (18.2%), Microsporium canis 2 (18.2%), C. albicans 2 (18.2%), Trichophyton rubrum 1 (9.1%), C. tropicalis 1 (9.1%). T. orientale isolates were collected from the gardens soil of the University of Baghdad and were then identified depending on the morphological feature of the colony and microscopic characteristics. To confirm identification, PCR technique was used in which DNA of T. orientale was extracted at a concentration of 54 - 294 μg/2 g wet mycelium with a purity of 1.6 - 1.8. DNA samples were amplified with primers ITS-1and ITS-4, And the results confirmed that the isolate was T. orientale strain FMR12486. This isolate was processed with ethyl acetate using a standard extraction method and the final amount of crude extract being 6 grams. Different concentrations of crude extract 0.5, 1, 2 and 4 mg/ml were prepared and used against fungal pathogens isolated using the agar-well diffusion method. The results showed high antifungal activity against pathogenic isolates, significantly recording maximum inhibition zones of 20 and 22 mm against C. parapsilosis at concentrations of 0.5 and 1 mg/ml, respectively. It was followed by T. mentagrophyte and C. glabrata. 16, 20, and 16, 20 mm that were recorded at concentrations of 2 and 4mg/ml, a significant maximum inhibition zone was recorded 26 mm against C. glabrata, followed by C. parapsilosis, M. canis and T. mentagrophyte (24, 26; 16, 24 and 20, 24mm respectively). Just Trichophyton rubrum fungus in this study showed resistance to all concentrations of the crude extract where the inhibition zone was zero. The results of the current study showed that T. orientale strain FMR12486 crude extract can be used to make an effective drug to treat human fungal infections.