Fungal Assays Research Articles

Improved Broad Spectrum Antifungal Drug Synergies with Cryptomycin, a Cdc50-Inspired Antifungal Peptide.

Fungal infections in humans are difficult to treat, with very limited drug options. Due to a confluence of factors, there is an urgent need for innovation in the antifungal drug space, particularly to combat increasing antifungal drug resistance. Our previous studies showed that Cdc50, a subunit of fungal lipid translocase (flippase), is essential for Cryptococcus neoformans virulence and required for antifungal drug resistance, suggesting that fungal lipid flippase could be a novel drug target. Here, we characterized an antifungal peptide, Cryptomycinamide (KKOO-NH2), derived from a 9-amino acid segment of the C. neoformans Cdc50 protein. A fungal killing assay indicated that KKOO-NH2 is fungicidal against C. neoformans. The peptide has antifungal activity against multiple major fungal pathogens with a minimum inhibitory concentration (MIC) of 8 μg/mL against C. neoformans and Candida glabrata, 16 μg/mL against Candida albicans and C. auris, and 32 μg/mL against Aspergillus fumigatus. The peptide has low cytotoxicity against host cells based on our hemolysis assays and vesicle leakage assays. Strikingly, the peptide exhibits strong drug synergy with multiple antifungal drugs, including amphotericin B, itraconazole, and caspofungin, depending on the specific species on which the combinations were assayed. The fluorescently labeled peptide was detected to localize to the plasma membrane, likely inhibiting key interactions of Cdc50 with membrane proteins such as P4 ATPases. Cryptococcus cells exposed to sub-MIC of peptide showed increased reactive oxygen species production and intracellular calcium levels, indicating a peptide-induced stress response. Decreased intracellular proliferation within macrophages was observed after 30 min of peptide exposure and 24 h coincubation with macrophages, providing a potential translational mechanism to explore further in vivo. In aggregate, the synergistic activity of our KKOO-NH2 peptide may offer a potential novel candidate for combination therapy with existing antifungal drugs.

Adhesion of Candida albicans on preformed crowns used to restore primary molars: An in vitro study

BackgroundAssessing microbial biofilm adhesion to the restorative material’s surface is a key factor in determining its success. This study aimed to compare the adhesion of Candida albicans (C. albicans) on stainless steel crowns (SSCs) versus zirconia crowns (ZCs) used for primary molars. Study designThis in vitro study involved 20 full-coverage crowns, with 10 SSCs and 10 ZCs. A fungal adhesion assay was performed to evaluate C. albicans adhesion, using colony-forming units (CFU) and cell proliferation assays. StatisticsThe Wilcoxon–Mann–Whitney test was applied to compare the groups, with statistical significance set at 5 %. ResultsThe study revealed that SSCs had a lower mean CFU value (28.28 ± 4.557 CFU/ml) and cell proliferation rate (49.74 ± 0.4367) compared to the ZC group (30.70 ± 11.0760 CFU/ml and 74.53 ± 0.7014, respectively). However, these differences were not statistically significant. ConclusionThe findings suggest that the selection of crowns for restoring primary molars may not be influenced by C. albicans adhesion.

Impact of tps1 Deletion and Overexpression on Terpene Metabolites in Trichoderma atroviride.

Terpenoids are structurally diverse natural products that have been widely used in the pharmaceutical, food, and cosmetic industries. Research has shown that fungi produce a variety of terpenoids, yet fungal terpene synthases remain not thoroughly explored. In this study, the tps1 gene, a crucial component of the terpene synthetic pathway, was isolated from Trichoderma atroviride HB20111 through genome mining. The function of this gene in the terpene synthetic pathway was investigated by constructing tps1-gene-deletion- and overexpression-engineered strains and evaluating the expression differences in the tps1 gene at the transcript level. HS-SPME-GC-MS analysis revealed significant variations in terpene metabolites among wild-type, tps1-deleted (Δtps1), and tps1-overexpressed (Otps1) strains; for instance, most sesquiterpene volatile organic compounds (VOCs) were notably reduced or absent in the Δtps1 strain, while nerolidol, β-acorenol, and guaiene were particularly produced by the Otps1 strain. However, both the Δtps1 and Otps1 strains produced new terpene metabolites compared to the wild-type, which indicated that the tps1 gene played an important role in terpene synthesis but was not the only gene involved in T. atroviride HB20111. The TPS1 protein encoded by the tps1 gene could function as a sesquiterpene cyclase through biological information and evolutionary tree analysis. Additionally, fungal inhibition assay and wheat growth promotion assay results suggested that the deletion or overexpression of the tps1 gene had a minimal impact on fungal inhibitory activity, plant growth promotion, and development, as well as stress response. This implies that these activities of T. atroviride HB20111 might result from a combination of multiple metabolites rather than being solely dependent on one specific metabolite. This study offers theoretical guidance for future investigations into the mechanism of terpenoid synthesis and serves as a foundation for related studies on terpenoid metabolic pathways in fungi.

Real-time application of ITS and D1-D3 nanopore amplicon metagenomic sequencing in fungal infections: Enhancing fungal infection diagnostics

While fungal infections cause considerable morbidity and mortality, the performance of the current diagnostic tests for fungal infection is low. Even though fungal metagenomics or targeted next-generation sequencing have been investigated for various clinical samples, the real-time clinical utility of these methods still needs to be elucidated. In this study, we used internal transcribed spacer (ITS) and D1-D3 ribosomal DNA nanopore amplicon metagenomic sequencing to assess its utility in patients with fungal infections. Eighty-four samples from seventy-three patients were included and categorized into ‘Fungal infection,’ ‘Fungal colonization,’ and ‘Fungal contamination’ groups based on the judgement of infectious disease specialists. In the ‘Fungal infection’ group, forty-seven initial samples were obtained from forty-seven patients. Three fungal cases detected not by the sequencing but by conventional fungal assays were excluded from the analysis. In the remaining cases, the conventional fungal assay-negative/sequencing-positive group (n=11) and conventional fungal assay-positive/sequencing-positive group (n=33) were compared. Non-Candida and non-Aspergillus fungi infections were more frequent in the conventional-negative/sequencing-positive group (p-value = 0.031). We demonstrated the presence of rare human pathogens, such as Trichosporon asahii and Phycomyces blakesleeanus. In the ‘Fungal infection’ group and ‘Fungal colonization’ group, sequencing was faster than culturing (mean difference = 4.92 days, p-value < 0.001/ mean difference = 4.67, p-value <0.001). Compared to the conventional diagnostic methods including culture, nanopore amplicon sequencing showed a shorter turnaround time and a higher detection rate for uncommon fungal pathogens.

Generic viability qPCR for monitoring shelf life of microbial biological control agents coated on seeds based on the nucleic acid intercalating dyes EMA and PMAxx

Biological control of seedborne pathogens and soilborne seedling pathogens through antagonists applied on seeds is an alternative to chemical seed treatments. Information on the viability of inocula on treated seeds is essential for any development and use of beneficial fungi or bacteria on seeds. Generic fungal and bacterial qPCR assays were combined with the nucleic acid intercalating dyes ethidium monoazide (EMA) and propidium monoazide (PMAxx) for the quantification of viable cells of fungi and bacteria on seeds. The applied protocols for generic fungal viability qPCR (v-qPCR) in combination with EMA and PMAxx and for generic bacterial v-qPCR in combination with PMAxx allowed the viability quantification of fungal and bacterial isolates representing a broad range of species with the exception of fungal species with highly melanized conidia. A first application of v-qPCR to coated seeds of onion and spinach indicated a differential plant species effect on survival of a coated fungus and a yeast with a generally better survival on seeds of spinach compared to seeds of onions and a similar good survival of the bacterium L. enzymogenes 3.1T8 on both seed types. The v-qPCR protocols can be applied in screening assays aiming at the selection of new antagonists with higher survival potentials and the development of new seed processing technologies compatible with coated antagonists.

Antagonistic activity of biocontrol agent Trichoderma spp. against Fusarium sp., the causal agent of Ananas comosus fruitlet rot

Pineapple (Ananas comosus) is a significant crop, with an annual production exceeding 25 million tons. However, fusariosis can severely impact its cultivation, a fungal disease that causes fruitlet rot and results in substantial yield losses. To decrease dependency on chemical control methods, biocontrol agents (BCAs) present a promising alternative. Among these, Trichoderma species are noteworthy due to their diverse antagonistic mechanisms. The efficacy of each mechanism can be assessed through fungal confrontation assays. This study aimed to isolate, identify, and evaluate in-vitro nine Trichoderma spp. strains as potential BCAs against Fusarium sp. associated with pineapple fruitlet rot. The antagonistic fungi were isolated from rhizosphere soils in both open-field and greenhouse pineapple farms in Misiones province, Argentina. Identification of the fungi required both morphologic and genetic data. In the in-vitro assays, the capabilities for direct competition for substratum, production of metabolites, and mycoparasitism were evaluated. The results indicated that isolates T. harzianum TC7, T. harzianum TC9, T. asperellum TU3, and T. asperellum TU4 had statistically superior inhibitory effects against Fusarium sp. These isolates can be potentially used in formulating natural fungicides to reduce pineapple fruitlet rot caused by Fusarium, promoting sustainable production practices. Keywords: pineapple, confrontation, mycoparasitism, metabolites, ITS region

Antagonistic activity of biocontrol agent Trichoderma spp. against Fusarium sp., the causal agent of Ananas comosus fruitlet rot

Pineapple (Ananas comosus) is a significant crop, with an annual production exceeding 25 million tons. However, fusariosis can severely impact its cultivation, a fungal disease that causes fruitlet rot and results in substantial yield losses. To decrease dependency on chemical control methods, biocontrol agents (BCAs) present a promising alternative. Among these, Trichoderma species are noteworthy due to their diverse antagonistic mechanisms. The efficacy of each mechanism can be assessed through fungal confrontation assays. This study aimed to isolate, identify, and evaluate in-vitro nine Trichoderma spp. strains as potential BCAs against Fusarium sp. associated with pineapple fruitlet rot. The antagonistic fungi were isolated from rhizosphere soils in both open-field and greenhouse pineapple farms in Misiones province, Argentina. Identification of the fungi required both morphologic and genetic data. In the in-vitro assays, the capabilities for direct competition for substratum, production of metabolites, and mycoparasitism were evaluated. The results indicated that isolates T. harzianum TC7, T. harzianum TC9, T. asperellum TU3, and T. asperellum TU4 had statistically superior inhibitory effects against Fusarium sp. These isolates can be potentially used in formulating natural fungicides to reduce pineapple fruitlet rot caused by Fusarium, promoting sustainable production practices. Keywords: pineapple, confrontation, mycoparasitism, metabolites, ITS region

Laser-disintegrated copper-activated salicylic acid (Cu-SA) nanoparticles suppressed the seed-borne pathogens of rice with enhanced seed invigoration parameters.

Leading phytopathological research is focused on managing seed-borne pathogens of rice through the utilization of engineered nanomaterials. Herein, blue laser-induced topo-morphologically nano-advanced copper salicylates (Cu-SA) (Cu/SA in 1:1 and 1:2 ratio) were prepared and evaluated for their augmented antifungal potential along seed invigoration effects in contrast to their prepared sonicated formulations. Laser disintegration on the Cu-SA (Cu/SA in 1:1 and 1:2 ratio) was achieved with high degree of success and precision using blue laser, which yielded uniformly distributed spherical nanoparticles with a narrow size distribution and better crystallinity than aqua-dispersed sonicated formulations. In vitro antifungal evaluation against seed-borne fungi of rice viz. Fusarium verticillioides and Fusarium fujikuroi revealed multiple times the augmented potential of laser-disintegrated nanoformulations (l-CuSA) than sonicated (s-CuSA) and bulk samples. Laser-induced nano-sodium bis(2-oxobenzoato)cuprate (II) (l-CuSA2) with Cu/SA in 1:2 ratio was the best to inhibit the in vitro fungal growth. Ultra-micrographs and fungal double-staining assay further rationalized the membrane disruption as the mode of action for the fungitoxicity. Nanopriming of fungal infested rice seeds with l-CuSA2 at 2500 μg/mL for 8 h showed the maximum reduction of seed rot (80.43%) and seedling blight (63.15%) with respect to control (untreated). The seed-invigorating factors of l-CuSA2 nanoprimed seeds were enhanced to maximum extent and showed the highest per cent germination (35.29%), shoot length (11.42%), root length (21.14%), dry weight (75.43%) and vigour index (81.04%) over the control. Inclusively, the seed nanopriming with l-CuSA2 proved as agro-compatible hypo-toxic semi natural nanoplatform for sustainable agriculture.

Development of a novel mycobiome diagnostic for fungal infection

BackgroundAmplicon-based mycobiome analysis has the potential to identify all fungal species within a sample and hence could provide a valuable diagnostic assay for use in clinical mycology settings. In the last decade, the mycobiome has been increasingly characterised by targeting the internal transcribed spacer (ITS) regions. Although ITS targets give broad coverage and high sensitivity, they fail to provide accurate quantitation as the copy number of ITS regions in fungal genomes is highly variable even within species. To address these issues, this study aimed to develop a novel NGS fungal diagnostic assay using an alternative amplicon target.MethodsNovel universal primers were designed to amplify a highly diverse single copy and uniformly sized DNA target (Tef1) to enable mycobiome analysis on the Illumina iSeq100 which is a low cost, small footprint and simple to use next-generation sequencing platform. To enable automated analysis and rapid results, a streamlined bioinformatics workflow and sequence database were also developed. Sequencing of mock fungal communities was performed to compare the Tef1 assay and established ITS1-based method. The assay was further evaluated using clinical respiratory samples and the feasibility of using internal spike-in quantitative controls was assessed.ResultsThe Tef1 assay successfully identified and quantified Aspergillus, Penicillium, Candida, Cryptococcus, Rhizopus, Fusarium and Lomentospora species from mock communities. The Tef1 assay was also capable of differentiating closely related species such as A. fumigatus and A. fischeri. In addition, it outperformed ITS1 at identifying A. fumigatus and other filamentous pathogens in mixed fungal communities (in the presence or absence of background human DNA). The assay could detect as few as 2 haploid genome equivalents of A. fumigatus from clinical respiratory samples. Lastly, spike-in controls were demonstrated to enable semi-quantitation of A. fumigatus load in clinical respiratory samples using sequencing data.ConclusionsThis study has developed and tested a novel metabarcoding target and found the assay outperforms ITS1 at identifying clinically relevant filamentous fungi. The assay is a promising diagnostic candidate that could provide affordable NGS analysis to clinical mycology laboratories.

REMOVAL OF Cr (VI) from NORKRAN’S LIQUID MEDIUM USING Aspergillus fumigatus and Rhizopus sp.

Soil and water pollution caused by heavy metals can result in health challenges such as cancer. Therefore, this study was aimed at testing the ability of Aspergillus fumigatus and Rhizopus sp. isolated from cassava grinding mill environment to reduce, bio-accumulate and tolerate Cr (VI) in Norkran’s medium. Fungal isolates were obtained from soil samples using 2 % malt extract agar. Cr (VI) reduction potential of the fungal cells was done using shake flask method. The tolerance of fungal isolates to the different concentrations of Cr (VI) used for the bio-sorption experiment was determined by collecting 1 ml of Norkran’s sample from each experimental flask on days 0, 5, 10 and 20 followed by inoculation in 2 % malt extract agar plates. The ability of fungal isolates to adapt to increasing Cr (VI) concentrations was ascertained by inoculating mycelial ball on 2 % malt extract agar amended with 16.1 mg/L of Cr (VI). There were reductions of Cr (VI) for all the treatments amended with A. fumigatus and Rhizopus sp. at 16.1, 8.1, 4.0 and 2.0 mg/L concentrations of Cr (VI) while 50 % decrease in Cr (VI) concentrations was observed for treatments inoculated with Rhizopus sp. on days 20, 10 and 5 at concentrations of 8.1, 4.0 and 2.0 mg/L respectively. The amounts of Cr (VI) observed at a concentration of 16.1 mg/L on day 20 for treatments inoculated with Rhizopus sp. were observed to be significantly less than (P&lt;0.05) those of A. fumigatus. The fungal tolerance assay demonstrated increase in fungal abundance throughout the experimental period for all the treatments indicating that the different concentrations of Cr (VI) used were not toxic to the fungal species. Induce-tolerance assay suggested that Cr (VI) was able to induce metal tolerance potential in these fungal species. Findings from this investigation revealed that these fungi can be further explored biotechnologically to clean-up chromium contaminated environment

Cross-sectional investigation of mycological diagnosis challenges in Saudi Arabia.

The global incidence of fungal infection has increased dramatically over the last two decades. Fungal diseases threaten both immunocompetent, and immunocompromised patients. The current fungal diagnostics status in Saudi Arabia needs to be evaluated, especially with the increase of the immunosuppressed population. This cross-sectional study investigated the gaps in mycological diagnosis on a national level. The call interview questionnaire responses were collected to evaluate the demand for fungal assays, diagnostic methods' quality, and mycological expertise of laboratory technologists in both public and private medical intuitions. The data were analyzed using (IBM SPSS ® software version 22.0). A total of 57 hospitals from all Saudi regions participated in the questionnaire; however, only 32% received or processed mycological samples. Most participants were from the Mecca region (25%), Riyadh region (19%), and Eastern region (14%). The top fungal isolates identified were Candida spp., Aspergillus spp., and dermatophyte. Fungal investigation is highly requested by intensive care, dermatology, and obstetrics and gynecology units. Most laboratories rely on fungal culture and microscopic examination, which mostly identify Candida to the genus level, and use 37°C incubators for culture (67%). Antifungal susceptibility testing (AST) and serological and molecular methods are rarely performed and mostly outsourced. Using accurate identification and AST are the primary factors to improve fungal diagnosis in respect to turnaround time and cost. The three major obstacles identified were availability of facility (47%), reagents and kits (32%), and good training (21%). The results indicated that fungal diagnosis demand was relatively higher in high-population regions. This study highlighted the gaps in fungal diagnostics reference laboratories to encourage their improvement in Saudi hospitals.

Knockdown of Bmp1 and Pls1 Virulence Genes by Exogenous Application of RNAi-Inducing dsRNA in Botrytis cinerea

Botrytis cinerea is a pathogen of wide agronomic and scientific importance partly due to its tendency to develop fungicide resistance. Recently, there has been great interest in the use of RNA interference as a control strategy against B. cinerea. In order to reduce the possible effects on non-target species, the sequence-dependent nature of RNAi can be used as an advantage to customize the design of dsRNA molecules. We selected two genes related to virulence: BcBmp1 (a MAP kinase essential for fungal pathogenesis) and BcPls1 (a tetraspanin related to appressorium penetration). After performing a prediction analysis of small interfering RNAs, dsRNAs of 344 (BcBmp1) and 413 (BcPls1) nucleotides were synthesized in vitro. We tested the effect of topical applications of dsRNAs, both in vitro by a fungal growth assay in microtiter plates and in vivo on artificially inoculated detached lettuce leaves. In both cases, topical applications of dsRNA led to gene knockdown with a delay in conidial germination for BcBmp1, an evident growth retardation for BcPls1, and a strong reduction in necrotic lesions on lettuce leaves for both genes. Furthermore, a strongly reduced expression of the BcBmp1 and BcPls1 genes was observed in both in vitro and in vivo experiments, suggesting that these genes could be promising targets for the development of RNAi-based fungicides against B. cinerea.

Candida albicans causes brain regional invasion and necrosis, and activation of microglia during lethal neonatal neurocandidiasis

Neurocandidiasis is a fungal infection that primarily affects neonates, which is associated with 70% case fatality rates, while pediatric patients who survive infection often have long-term neurological sequelae, making it a clinical requirement to understand the pathogenesis of neonatal neurocandidiasis. Currently, the brain regions to Candida albicans invasion during the neonatal period are not characterized. In this study, 0-day-old mice were infected with C. albicans intravenously to determine dissemination and invasion into the brain at different times post-infection by fungal burden assay and histopathological analysis, additionally cellular death and microglial activation were evaluated by flow cytometry. The results evidenced the dissemination of C. albicans within the first hour of infection in the brain. The meninges were the initial site of invasion during the first 6 hours post infection and then filamentous structures into the brain parenchyma increases during infection, the anatomic regions most susceptible to invasion being the cerebral cortex, thalamus, hypothalamus, midbrain, pons, and medulla oblongata. Furthermore, C. albicans invasion of brain tissue results in cell necrosis and activation of microglia as a consequence of fungal invasion.

Antibiofilm Activity of 3D-Printed Nanocomposite Resin: Impact of ZrO2 Nanoparticles.

Poly(methyl methacrylate) (PMMA) is a commonly used material, as it is biocompatible and relatively cheap. However, its mechanical properties and weak antibiofilm activity are major concerns. With the development of new technology, 3D-printed resins are emerging as replacements for PMMA. Few studies have investigated the antibiofilm activity of 3D-printed resins. Therefore, this study aimed to investigate the antibiofilm activity and surface roughness of a 3D-printed denture base resin modified with different concentrations of zirconium dioxide nanoparticles (ZrO2 NPs). A total of 60 resin disc specimens (15 × 2 mm) were fabricated and divided into six groups (n = 10). The groups comprised a heat-polymerized resin (PMMA) group, an unmodified 3D-printed resin (NextDent) group, and four 3D-printed resin groups that were modified with ZrO2 NPs at various concentrations (0.5 wt%, 1 wt%, 3 wt%, and 5 wt%). All specimens were polished using a conventional method and then placed in a thermocycler machine for 5000 cycles. Surface roughness (Ra, µm) was measured using a non-contact profilometer. The adhesion of Candida albicans (C. albicans) was measured using a fungal adhesion assay that consisted of a colony forming unit assay and a cell proliferation assay. The data were analyzed using Shapiro-Wilk and Kruskal-Wallis tests. A Mann-Whitney U test was used for pairwise comparison, and p-values of less than 0.05 were considered statistically significant. The lowest Ra value (0.88 ± 0.087 µm) was recorded for the PMMA group. In comparison to the PMMA group, the 3% ZrO2 NPs 3D-printed group showed a significant increase in Ra (p < 0.025). For the 3D-printed resins, significant differences were found between the groups with 0% vs. 3% ZrO2 NPs and 3% vs. 5% ZrO2 NPs (p < 0.025). The highest Ra value (0.96 ± 0.06 µm) was recorded for the 3% ZrO2 NPs group, and the lowest Ra values (0.91 ± 0.03 µm) were recorded for the 0.5% and 5% ZrO2 NPs groups. In terms of antifungal activity, the cell proliferation assay showed a significant decrease in the C. albicans count for the 0.5% ZrO2 NPs group when compared with PMMA and all other groups of 3D-printed resins. The group with the lowest concentration of ZrO2 NPs (0.5%) showed the lowest level of C. albicans adhesion of all the tested groups and showed the lowest Candida count (0.29 ± 0.03). The addition of ZrO2 NPs in low concentrations did not affect the surface roughness of the 3D-printed resins. These 3D-printed resins with low concentrations of nanocomposites could be used as possible materials for the prevention and treatment of denture stomatitis, due to their antibiofilm activities.

85. Plasma Cell Free Deoxyribonucleic Acid (cfDNA) Fungal Polymerase Chain Reaction (PCR): a Non-Invasive Diagnostic for Invasive Fungal Disease Evaluation in At-Risk Pediatric Oncology and Stem Cell Transplant Patients

Abstract Background Molecular diagnostics appear promising for early, non-invasive detection of invasive fungal disease (IFD) in immunocompromised patients. Our clinical lab developed and validated cell free DNA (cfDNA) fungal polymerase chain reaction (PCR) assays (Table 1), which have been in clinical use since November 2020 and were recently included in an institutional pediatric clinical care pathway for prolonged febrile neutropenia. We aimed to evaluate the performance of these plasma cfDNA fungal PCR assays in pediatric oncology and hematopoietic stem cell transplant (HSCT) patients with clinical concern for IFD. Methods We initiated an observational study of inpatient oncology and HSCT patients who had plasma mold panel (+/- Candida panel) cfDNA fungal PCR obtained as part of an IFD evaluation at our freestanding quaternary care children’s hospital. The primary outcome was IFD clinical diagnosis (proven, probable, possible, unlikely) as per EORTC/MSG† definitions within one month of the cfDNA fungal PCR, which was assigned independently by two physicians, with a third physician utilized in cases of discrepancy. Patient demographics, hospital course, imaging results, and clinical laboratory data were abstracted and maintained in an encrypted REDCap© database. Results In a preliminary analysis of October 2021-March 2022 data, there were 21 IFD evaluations for 18 patients (Table 2). Most oncology evaluations were for prolonged febrile neutropenia, while many HSCT were non-neutropenic, but on enhanced immunosuppression with new clinical concerns (e.g., respiratory symptoms, persistent fever). Plasma cfDNA detected a mold or yeast consistent with the clinical presentation in 100% of the five proven/probable cases (Figures 1 &amp; 2). All 14 possible IFD cases had a negative cfDNA fungal PCR. Proven cases are designated with blue icon, while the probable case is orange. All five cases were in oncology patients who did not have history of hematopoietic stem cell transplant. Cell free DNA (cfDNA) fungal polymerase chain reaction (PCR) results include the organism identified, followed by the cycle threshold (Ct) in parenthesis. Maximum Ct for the assay is 45. Abbreviations: AG: Aspergillus galactomannan index (Ref &amp;lt;0.5); ALL: acute lymphoblastic leukemia. BAL: bronchoalveolar lavage; 1,3-BDG: 1,3-beta-D-glucan (Ref &amp;lt;60 pg/ml); cfDNA: cell free deoxyribonucleic acid; CNS: central nervous system; EORTC/MSG: European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group &amp; the National Institute of Allergy &amp; Infectious Diseases Mycoses Study Group; L-AmB: liposomal amphotericin B; SARS-CoV-2: severe acute respiratory syndrome associated with coronavirus 2. Conclusion Upfront plasma cfDNA fungal PCR successfully detected a relevant mold or yeast with short turnaround time in 100% of cases that were later classified as proven/probable. This appears promising for early, noninvasive diagnosis that enables prompt optimization of antifungal and surgical management, particularly for mucormycosis cases. Additional data may permit consideration of Mucorales agents PCR as a new EORTC/MSG mycologic criteria. Disclosures Lauren E. Kushner, MD, Roman: Client of my husband's business..

Evaluation of the Diagnostic Accuracy and Clinical Utility of Fungal Profile Plus Polymerase Chain Reaction Assay in Pulmonary Infections.

Pulmonary infections due to Aspergillus, Mucorales, and Nocardia have high morbidity and mortality, in part due to diagnostic challenges. Commercially available molecular assays on bronchoalveolar lavage fluid (BALF) may have increased sensitivity over currently available diagnostic options. Our aim was to characterize the diagnostic performance of assays for each of these pathogens in our patient population. The medical records of patients whose BALF was tested by polymerase chain reaction (PCR) for Aspergillus, Mucorales, and Nocardia between 2019 and 2021 were reviewed in a cross-sectional manner. European Organization for Research and Treatment of Cancer and the Mycoses Study Group (EORTC/MSG) definitions of "proven," "probable," and "possible" infection were used, including histopathology, serology, and culture. We used (1) "proven" or "probable" infection by EORTC criteria, (2) improvement or stabilization on targeted antimicrobial therapy, and (3) absence of a more likely diagnosis as the reference standard. The Aspergillus PCR assay demonstrated the highest agreement with the diagnostic reference standard, with 31.25% (10/32) sensitivity and 97.17% (206/212) specificity. Positive and negative predictive values were 62.50% (10/16) and 90.35% (206/228), respectively. No Mucorales or Nocardia infections were identified by the diagnostic reference standard, so the sensitivity could not be calculated. The specificity of Mucorales and Nocardia targets was 98.35% and 96.69%, respectively. Our data demonstrated relatively poor clinical sensitivity for all 3 constituent PCR assays in our patient population, suggesting a limited role for this test in the diagnosis of Aspergillus, Mucorales, or Nocardia.

Gallic Acid Ameliorates Aspergillus Fumigatus Keratitis Through Reducing Fungal Load and Suppressing the Inflammatory Response.

The purpose of this study was to explore the antifungal and anti-inflammatory effects of gallic acid (GA) on Aspergillus fumigatus (A. fumigatus) keratitis. CCK-8 assay and Draize eye test were used to determine the non-cytotoxic concentration of GA in RAW264.7 cells and an A. fumigatus keratitis mouse model. The antifungal effects of GA were analyzed using minimal inhibitory concentration (MIC), biofilm formation test, fungal adherence assay, calcofluor white staining, and propidium iodide staining. The therapeutic effects of GA were estimated by slit lamp photographs, clinical score, hematoxylin and eosin (H&E) staining, and Periodic acid-Schiff staining in vivo. Immunofluorescence staining and myeloperoxidase assay were conducted to identify neutrophil infiltration and activity. RT-PCR, ELISA, and Western blot were performed to detect the expression of pro-inflammatory cytokines and Nrf2/HO-1. In HCECs and A. fumigatus keratitis mouse model, GA at 100 µg/mL did not affect cell viability, thus this concentration was applied to subsequent experiments. In vitro, GA significantly inhibited A. fumigatus growth, biofilm formation, and adhesion. In vivo, 100 µg/mL GA alleviated the severity of fungal keratitis (FK) by repressing fungal load, reducing neutrophil infiltration, and lowering MPO activity. Besides, the expression of IL-1β, TNF-α, LOX-1, and COX-2 was inhibited, whereas Nrf2 and HO-1 expression was enhanced at both mRNA and protein levels in the 100 µg/mL GA treated group in comparison to PBS control. GA ameliorates FK severity through inhibiting A. fumigatus load, reducing neutrophils infiltration, downregulating the expression of pro-inflammatory cytokines, and enhancing the Nrf2/HO-1 pathway, which provides new insight into A. fumigatus keratitis treatment.

Comparison of Indirect Fungal Diagnostic Tests in Patients With Proven Histoplasmosis.

Histoplasmosis is a common cause of invasive fungal infection in endemic regions and accurate diagnosis is difficult without direct tissue culture or pathology. Indirect fungal antigen testing for various fungal pathogens are typically performed to assist with diagnostic workup, though cross-reaction can lead to difficulty in interpreting results. We aimed to compare indirect fungal diagnostic tests and evaluate prevalence of positive antigen testing for non-Histoplasma fungal pathogens in patients with proven histoplasmosis. We performed a single-center retrospective review of adult patients with proven histoplasmosis diagnosed by fungal culture and/or cytology from January 2010 to March 2018. Patient demographics, clinical characteristics, and results of fungal antigen testing for Histoplasma, Blastomyces, Aspergillus, Cryptococcus, and (1→3)-β-D-glucan were evaluated. Two different urine Histoplasma antigen assays were used during the study period. Fifty-seven of 182 (31.3%) patients reviewed had proven histoplasmosis and presented with acute pulmonary (n = 10), chronic pulmonary (n = 7), and disseminated (n = 40) disease. Forty-one (72%) of these patients were immunosuppressed. Urine Blastomyces antigen (93%) and serum (1→3)-β-D-glucan (88%) were commonly positive in patients with histoplasmosis, whereas Aspergillus antigen was detected in 50% of patients and Cryptococcus antigenemia was rare (5%). In patients with disseminated disease, the MiraVista urine Histoplasma antigen assay had higher sensitivity than the Viracor urine Histoplasma antigen assay (86% vs 50%, respectively; P = .019). Noninvasive fungal antigen assays are helpful diagnostic tools; however, given their low specificity, clinicians must be aware of the various clinical presentations of invasive fungal infections and be aware of the limitations of these tests.

The nature of the fungal cargo induces significantly different temporal programmes of macrophage phagocytosis

Phagocytosis is an essential component of our immune defence against fungal pathogens. Differences in the dynamics of phagocyte migration, recognition, uptake and phagolysosome maturation are dependent on the characteristics of the fungal cargo, and in particular to differences in cell wall composition and cellular morphology. However, studies that have focused on phagocyte interactions with individual fungal species have not enabled comparisons in the kinetics of these interactions to be made between these different species. We therefore used live cell video microscopy to examine the temporal dynamics of phagocytosis for a range of fungal cargoes by thioglycollate–elicited peritoneal macrophages from C57BL/6 mice. Uniform populations of macrophages were challenged at the same time with yeast cells of Candida albicans, Candida glabrata, Saccharomyces cerevisiae and Cryptococcus neoformans (wild–type and an acapsular mutant, cap59Δ), and spores of Aspergillus fumigatus and Mucor circinelloides to enable standardized comparative interactions to be quantified from different stages of phagocytosis. Differences in the rate of uptake of fungal cells varied by up to 26–fold, whilst differences in time to induce phagosome acidification varied by as much as 29–fold. Heat-killing or opsonizing the fungal targets markedly affected the kinetics of the interaction in a species–specific manner. Fungal and macrophage killing assays further revealed cargo–specific differences in phagocytosis and diversity in fungal evasion mechanisms. Therefore, simultaneous assessment of the interaction of macrophages with different fungal pathogens highlighted major differences in the kinetics and growth responses during fungus–phagocyte interactions that are likely to impact on pathogenesis and virulence.

Modular, multi‐barcode amplicon sequencing for improved species‐level detection of fungal phytopathogens: A case study of pipeline establishment targeting the Ophiostomatales

AbstractRecent advances in the field of environmental DNA (eDNA) metabarcoding have produced a promising platform for early detection and broad‐spectrum monitoring of plant pests and pathogens. To date, the majority of fungal metabarcoding assays have relied upon universal primers amplifying segments of the ribosomal DNA, to ensure broad taxonomic coverage while studying changes in community composition, or more recently, to screen for presence/absence of target species. In a diagnostics framework however, single, universal molecular barcodes do not discriminate accurately enough for many groups of important fungal phytopathogens at the required species level. Here, a modular, multi‐barcode amplicon sequencing pipeline was established to provide rapid and reliable diagnostics targeting the Ophiostomatales, an order containing economically important phytopathogens vectored by bark and ambrosia beetles (Curculionidae). Using compositionally varied mock communities, we evaluated five barcoding loci: ITS1, ITS2, the large ribosomal sub‐unit (LSU), translation elongation factor 1‐alpha (TEF1α) and calmodulin (CAL), for their ability to provide species‐level resolution. The sensitivity and detection limit of the assay was established by spiking genomic DNA of the exotic Dutch elm disease pathogen, Ophiostoma novo‐ulmi Brasier, into both the controlled mock communities and eDNA sampled from Ips grandicollis (Eichhoff) beetles collected in New South Wales, Australia. The TEF1α barcode successfully detected the Dutch elm pathogen DNA with 100% accuracy down to approx. 5 pg/μl, while the ITS1 barcode had a 100% accuracy down to approx. 0.4 pg/μl within a given sample. Our results demonstrate that a dual‐barcode approach targeting the ITS1 and TEF1α regions simultaneously is sufficient for accurate species level detection and characterisation of the Ophiostomatales and offers confident detection of low‐abundance taxa (relative read abundances of 0.1%–0.01%). Multi‐barcode metabarcoding provides a framework which can quickly and efficiently be adapted to new targets when establishing high throughput diagnostics for post‐border biosecurity surveillance of fungal phytopathogens.