Calcofluor White Staining Research Articles

Novel Essential Oil-Based Thiosemicarbazone Compounds as Potential Fungicides in Controlling Postharvest Diseases of Citrus Fruit.

To develop novel fungicides for controlling postharvest fungal diseases in citrus fruits, 12 essential oil (EO)-based thiosemicarbazones compounds, termed hydrazine-carbothioamide, were prepared according to the condensation method. In vitro assays showed that compound 13j exhibited the strongest antifungal activity (minimum inhibitory concentration [MIC] = minimum fungicidal concentration [MFC] = 0.0125 mg/mL) against Penicillium digitatum. An in vivo study revealed that 5 × MFC of compound 13j can effectively mitigate the green mold incidence of citrus fruit inoculated with P. digitatum, as well as fruit rot during natural storage, at a level comparable to that of the chemical fungicide prochloraz. Throughout this process, fruit quality was maintained. The hemolysis assay showed that these thiosemicarbazone compounds have good biocompatibility and that their safety is comparable to that of prochloraz. The antifungal activity of compound 13j was attributed to membrane damage, as confirmed using scanning electron microscopy (SEM), Calcofluor white (CFW) staining, propidium iodide (PI) staining, Fourier transform-infrared (FT-IR) spectroscopy, optical density (OD)260, and relative conductivity assays. Collectively, our results indicate that compound 13j can be used as an antifungal agent to control the postharvest decay of citrus fruits.

Clinico-Mycological Study of Dermatophytosis among Diabetic and Non-Diabetic Patients in a Tertiary Level Hospital: A Comparative Study

Abstract Background: Dermatophytosis is a major public health concern in India, especially in recent years, with an alarmingly rising trend, particularly in relation to recurrent and chronic infection. The number of studies examining the relationship between an individual’s glycemic status and the development of dermatophytosis, and a comparison of the evolving species trend between diabetic and non-diabetic patients infected with dermatophytes, is limited. Aims and Objectives: To study and compare the clinical and mycological aspects of dermatophytosis among diabetic and non-diabetic patients and to compare the culture isolates in both groups. Materials and Methods: The study included 378 patients of clinically suspected dermatophytosis, divided into two groups of 189 known diabetics and non-diabetics each. We subsequently analysed and compared the demographics, clinical data, potassium hydroxide (KOH) mount, calcofluor white (CW) staining and fungal culture results of all patients in both groups. Results: Among the 378 patients, diabetic patients had a significantly greater extent of involvement and higher rates of recurrence when compared to non-diabetics. The overall positivity rate was higher in CW staining (77.8%) as compared to KOH (57.7%) in both non-diabetics and diabetics. Trichophyton mentagrophytes was the most common isolate (47.6%) in both diabetic (55.0%) and non-diabetic (61.4%) patients, followed by Trichophyton rubrum (31.8% and 29.6%, respectively). The positivity percentages of fungal culture, KOH and CW staining increased to 100%, 77.9% and 95.7%, respectively, when scrapings were taken from ≥2 sites. Conclusion: The pattern of dermatophytosis is comparatively more chronic and severe in diabetics compared to non-diabetics. T. mentagrophytes was the most common culture isolate in both groups. CW staining can potentially be used as the initial method of choice for the diagnosis of dermatophytosis as it has significantly outperformed the conventional KOH mount. Scrapings can regularly be taken from ≥2 sites to avoid false negative results.

Plant-derived citronellol can significantly disrupt cell wall integrity maintenance of Colletotrichum camelliae

Anthracnose, a fungal disease, commonly infects tea plants and severely impacts the yield and quality of tea. One method for controlling anthracnose is the application of citronellol, a plant extract that exhibits broad-spectrum antimicrobial activity. Herein, the physiological and biochemical mechanism by which citronellol controls anthracnose caused by Colletotrichum camelliae was investigated. Citronellol exhibited excellent antifungal activity based on direct and indirect mycelial growth inhibition assays, with EC50 values of 76.88 mg/L and 29.79 μL/L air, respectively. Citronellol also exhibited good control effects on C. camelliae in semi-isolated leaf experiments. Optical and scanning electron microscopy revealed that citronellol caused C. camelliae mycelia to thin, fracture, fold and deform. Transmission electron microscopy revealed that the mycelial cell walls collapsed inward and separated, and the organelles became blurred after treatment with citronellol. The sensitivity of C. camelliae to calcofluor white staining was significantly enhanced by citronellol, while PI staining showed minimal fluorescence, and the relative conductivity of mycelia were not significantly different. Under citronellol treatment, the expression levels of β-1,3-glucanase, chitin synthase, and chitin deacetylase-related genes were significantly decreased, while the expression levels of chitinase genes were increased, leading to lower chitinase activity and increased β-1,3-glucanase activity. Therefore, citronellol disrupted the cell wall integrity of C. camelliae and inhibited normal mycelial growth.

Combination of Cinnamaldehyde/β-cyclodextrin inclusion complex and L-phenylalanine effectively reduces the postharvest green mold in citrus fruit

The essential oil and β-cyclodextrin inclusion complex was able to inhibit the growth of Penicillium digitatum, a damaging pathogen that causes green mold in citrus fruit. In this study, cinnamaldehyde-β-cyclodextrin inclusion complex (β-CDCA) for controlling citrus green mold was synthesized by the co-precipitation method. Characterization of β-CDCA revealed that the aromatic ring skeleton of cinnamaldehyde (CA) was successfully embedded into the cavity of β-CD to form the inclusion complex. β-CDCA inhibited P. digitatum at a minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of 4.0 g/L. FT-IR spectroscopy analysis, calcofluor white staining, extracellular alkaline phosphatase (AKP) activity and propidium iodide (PI) staining of hyphae morphology showed that β-CDCA may damage the cell ultrastructure and membrane permeability of P. digitatum. The study further demonstrated that hydrogen peroxide (H2O2), malondialdehyde (MDA), and reactive oxygen species (ROS) markedly accumulated in 1/2 MIC β-CDCA treated hyphae. This implied that β-CDCA inhibited growth of P. digitatum by the triggering oxidative stress, which may have caused cell death by altering cell membrane permeability. In addition, in vivo results showed that β-CDCA alone or combined with L-phenylalanine (L-PHe) displayed a comparable level to that of prochloraz. Therefore, β-CDCA combined with L-PHe can thus be used as an eco-friendly preservative for the control green mold in postharvest citrus fruit.

Dermatophytosis in domestic cats: Identification, and treatment in an Indian context

The surge in domestic cat adoption across India, particularly the rising preference for high-pedigree cats, coupled with environmental factors, has resulted in increased incidence of dermatophytosis among feline companions. Despite this growing concern, there is a noticeable scarcity of studies in India delving into the etiological factors contributing to dermatophytosis in cats. This disease is a threat to animal health and carries public health significance, given that cats are recognized reservoir hosts for Microsporum canis, a common dermatophyte affecting humans and animals. This study endeavours to identify the dermatophytes affecting cats and establish a standardized therapeutic regimen while accounting for the local stigma surrounding the regular bathing of cats. The study involved the examination of 82 cats presenting dermatological lesions, when subjected to cultural examination in dermatophyte test medium revealed 36 afflicted with dermatophytes. Isolates were presumptively identified by staining using lactophenol cotton blue, Chicago sky blue 6B, and Calcofluor white stains. Molecular-level identification of the isolates was confirmed through PCR-RFLP, amplifying the Internal Transcribed Spacer Sequence of 16 s rDNA, followed by restriction digestion using the Mva1 enzyme. Among the thirty-six isolates, 29 were identified as M. canis, while the remaining 7 were M. gypseum. The cases were categorized into five groups and treated with Lime Sulphur dip, 4 % chlorhexidine shampoo, a shampoo containing 2 % miconazole and 4 % chlorhexidine, oral itraconazole alone, and a combination of oral itraconazole with lime-Sulphur dip. Statistical analysis revealed that the response was notably swifter with lime Sulphur dip when considering only topical therapy. Moreover, the mycological cure was most expeditious when combining Lime Sulphur dip with oral itraconazole. These findings underscore the pivotal role of topical biocides in feline dermatophytosis treatment, potentially reducing the reliance on specific antifungals and thereby contributing to the mitigation of antimicrobial resistance emergence.

Microbiological Profile of Culture-Positive Fungal Keratitis.

To examine the microbiological profile of cases of culture-positive fungal keratitis presenting to a tertiary eye care center in eastern India. Microbiology records of all culture-positive microbial keratitis patients presenting to L V Prasad Eye Institute, Bhubaneswar, between January 2020 and December 2021, were retrospectively reviewed. Collected data included smear results of culture-positive fungal or mixed infections, the species isolated, and the time taken for organisms to grow in each media. Fungal keratitis formed 36% of all culture-positive microbial keratitis, whereas mixed infections (fungi and other organisms) formed 8.5%. The most common fungal species isolated was Fusarium spp. (25.8%). The most common bacteria involved in mixed infection with fungi was Staphylococcus spp. (54.8%). The positivity of potassium hydroxide+calcofluor white stain in detecting fungal filaments was 89.0% and that of Gram stain was 76.1%. Culture-positive cases of fungal keratitis showed most frequent growth on potato-dextrose agar (77.6%). A similar pattern was observed in culture-positive mixed infections (Sabouraud dextrose agar [SDA]: 84%). Most frequent growth of bacteria in mixed infections was seen in thioglycolate broth (54.7%). The shortest time to achieve significant fungal growth was observed in blood agar (BA) and chocolate agar (CA) (2.2/2.3 days, and 1.8/2 days for fungal keratitis and mixed infections, respectively). Filamentous hyaline fungi took the shortest time to achieve significant growth (2.8 days), whereas yeast forms took the longest (5 days). This study highlights the importance of combined use of both solid and liquid culture media, especially potato dextrose agar (PDA)/SDA and CA, to arrive at a definitive diagnosis of fungal keratitis and possible bacterial co-infection, which forms a significant proportion of cases with fungal keratitis. In resource-poor laboratories, two culture media, either SDA or PDA, along with BA, may be plated to detect mixed infections. Examination of stained smears of corneal samples provides an inexpensive method of rapid diagnosis of fungal keratitis when culture media is not available.

Resveratrol has neuroprotective effects and plays an anti-inflammatory role through Dectin-1/p38 pathway in Aspergillus fumigatus keratitis

PurposeTo determine the antifungal, anti-inflammatory and neuroprotective effects of resveratrol (RES) in Aspergillus fumigatus (A. fumigatus) keratitis. MethodsCytotoxicity assay and Draize eye assay were performed to assess the toxicity of RES. The antifungal effect of RES was assessed by minimal inhibitory concentration, scanning or transmission electron microscopy, propidium iodide uptake assay, and Calcofluor white staining. Phosphorylation of p38 MAPK, mRNA and protein levels of Dectin-1 and related inflammatory factors were measured by qRT-PCR, ELISA and Western blot in vitro and in vivo. Clinical score, HE staining, plate count, and myeloperoxidase test were used to observe the progress of fungal keratitis. IF staining, qRT-PCR, and the Von Frey test were selected to assess the neuroprotective effects of RES. ResultsRES suppressed A. fumigatus hyphae growth and altered hyphae morphology in vitro. RES decreased the expression of Dectin-1, IL-1β and TNF-α, as well as p38 MAPK phosphorylation expression, and also decreased clinical scores, reduced inflammatory cell infiltration and neutrophil activity, and decreased fungal load. RES also protected corneal basal nerve fibers, down-regulated mechanosensitivity thresholds, and increased the mRNA levels of CGRP and TRPV-1.. ConclusionThese evidences revealed that RES could exert antifungal effects on A. fumigatus and ameliorate FK through suppressing the Dectin-1/p38 MAPK pathway to down-regulate IL-1β, IL-6, etc. expression and play protective effect on corneal nerves.

Melanin depletion affects Aspergillus flavus conidial surface proteins, architecture, and virulence.

Melanin is an Aspergillus flavus cell wall component that provides chemical and physical protection to the organism. However, the molecular and biological mechanisms modulating melanin-mediated host-pathogen interaction in A. flavus keratitis are not well understood. This work aimed to compare the morphology, surface proteome profile, and virulence of melanized conidia (MC) and non-melanized conidia (NMC) of A. flavus. Kojic acid treatment inhibited melanin synthesis in A. flavus, and the conidial surface protein profile was significantly different in kojic acid-treated non-melanized conidia. Several cell wall-associated proteins and proteins responsible for oxidative stress, carbohydrate, and chitin metabolic pathways were found only in the formic acid extracts of NMC. Scanning electron microscopy (SEM) analysis showed the conidial surface morphology difference between the NMC and MC, indicating the role of melanin in the structural integrity of the conidial cell wall. The levels of calcofluor white staining efficiency were different, but there was no microscopic morphology difference in lactophenol cotton blue staining between MC and NMC. Evaluation of the virulence of MC and NMC in the Galleria mellonella model showed NMC was less virulent compared to MC. Our findings showed that the integrity of the conidial surface is controlled by the melanin layer. The alteration in the surface protein profile indicated that many surface proteins are masked by the melanin layer, and hence, melanin can modulate the host response by preventing the exposure of fungal proteins to the host immune defense system. The G. mellonella virulence assay also confirmed that the NMC were susceptible to host defense as in other Aspergillus pathogens. KEY POINTS: • l-DOPA melanin production was inhibited in A. flavus isolates by kojic acid, and for the first time, scanning electron microscopy (SEM) analysis revealed morphological differences between MC and NMC of A. flavus strains • Proteome profile of non-melanized conidia showed more conidial surface proteins and these proteins were mainly involved in the virulence, oxidative stress, and metabolism pathways • Non-melanized conidia of A. flavus strains were shown to be less virulent than melanised conidia in an in vivo virulence experiment with the G. melonella model.

Synergistic antifungal mechanism of cinnamaldehyde and nonanal against Aspergillus flavus and its application in food preservation

Aspergillus flavus colonization on agricultural products during preharvest and postharvest results in tremendous economic losses. Inspired by the synergistic antifungal effects of essential oils, the aims of this study were to explore the mechanism of combined cinnamaldehyde and nonanal (SCAN) against A. flavus and to evaluate the antifungal activity of SCAN loading into diatomite (DM). Shriveled mycelia were observed by scanning electron microscopy, especially in the SCAN treatment group. Calcofluor white staining, transmission electron microscopy, dichloro-dihydro-fluorescein diacetate staining and the inhibition of key enzymes in tricarboxylic acid cycle indicated that the antifungal mechanism of SCAN against A. flavus was related to the cell wall damage, reactive oxygen species accumulation and energy metabolism interruption. RNA sequencing revealed that some genes involved in antioxidation were upregulated, whereas genes responsible for cell wall biosynthesis, oxidative stress, cell cycle and spore development were significantly downregulated, supporting the occurrence of cellular apoptosis. In addition, compared with the control group, conidia production in 1.5 mg/mL DM/cinnamaldehyde, DM/nonanal and DM/SCAN groups were decreased by 27.16%, 48.22% and 76.66%, respectively, and the aflatoxin B1 (AFB1) contents decreased by 2.00%, 73.02% and 84.15%, respectively. These finding suggest that DM/SCAN complex has potential uses in food preservation.

Examination of Oral Candida Infection in Primary Sjögren's Syndrome Patients

Primary Sjögren's syndrome (pSS) is an autoimmune disease characterized by symptoms such as dry mouth, dry eyes, and other systematic symptoms. Due to the hyposalivation experienced by pSS patients, oral dysbacteriosis often occurs. A common complication of pSS is the oral Candida infection. In this article, the authors describe systematic methods that can effectively diagnose oral Candida infection and identify the Candida strains using saliva, oral mucosal swabs, or mouthwash from pSS patients. The Sabouraud's Dextrose Agar (SDA), hyphal formation assay, potassium hydroxide (KOH) smear test, and calcofluor white (CFW) staining assay are used for the diagnosis of oral Candida infection. A Candida diagnostic agar is used for the identification of Candida strains. Finally, antifungal susceptibility testing is used to determine appropriate antifungal drug treatment. This standardized method can enhance the diagnosis, treatment, and future research of pSS-related oral Candida infections. Early diagnosis, using this method, can also prevent any complications arising due to delay in receiving appropriate treatment.

Diagnostic values of ten methods in patients with onychomycosis: A network meta‐analysis

AbstractBackgroundOnychomycosis, a disease was with a high global prevalence, has been the focal point of this study. The research aims to compare diagnostic efficacy among various methods, including potassium hydroxide (KOH), Dimethyl sulfoxide (DMSO), specific colour staining (Chlorazol, lactophenol cotton blue and Chicago sky blue stain), fluorescent staining (calcofluor white stain, acridine orange fluorescent dye), culture on dermatophyte test medium (DTM), conventional PCR, dermoscopy, histopathological examination using PAS stain (HEP‐PAS), real‐time PCR and multiple PCR. This comparison was achieved through a network meta‐analysis involving patients with onychomycosis.MethodsA comprehensive literature search was conducted across eight databases. Subsequently, both direct and indirect evidence were amalgamated to assess sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy. This combination analysis helped to estimate the odds ratios (ORs) and surface under the cumulative ranking curve (SUCRA) values of the nine different methods, providing an in‐depth perspective on diagnosing onychomycosis.ResultsThe network meta‐analysis integrated 20 eligible diagnostic trials. The results exhibited that DTM provided higher sensitivity, NPV and accuracy, as indicated by SUCRA values. Meanwhile, DMSO demonstrated higher specificity and positive predictive value in terms of SUCRA values. Among the nine methods, fluorescent staining showcased comparatively superior sensitivity, specificity, NPV, PPV and accuracy. Furthermore, a cluster analysis indicated that DTM held the highest diagnostic value for sensitivity and accuracy among the nine techniques, with fluorescence staining ranking second.ConclusionThis network meta‐analysis strengthens the evidence suggesting that fluorescence staining exhibits strong diagnostic value in onychomycosis. This finding bears diagnostic implications for onychomycosis.

The value of calcofluor white in the diagnosis of invasive fungal diseases

PurposeTo evaluate the value of calcofluor white in the diagnosis of invasive fungal disease (IFD). MethodsA total of 84 patients with possible pulmonary fungal infection who underwent bronchoscopy with bronchoalveolar lavage fluid (BALF) were included. All BALF specimens were subjected to Calcofluor white (CFW), potassium hydroxide (KOH) and Gram stains. ResultsCFW has the most sensitivity than KOH and Gram staining. The specificity of CFW was 92.00 %, which was lower than that of Gram staining. The PPVs for CFW, KOH and Gram staining were 94.44 %, 84.62 % and 80.00 % respectively. The NPVs for CFW, KOH and Gram staining was 47.92 %, 32.39 % and 30.38 % respectively. The AUCs of these three methods were 0.748, 0.550 and 0.510 respectively. ConclusionCFW is superior to KOH and Gram staining in the diagnosis of invasive fungal diseases.

Proteome and gut microbiota analysis of Chinese mitten crab (Eriocheir sinensis) in response to Hepatospora eriocheir infection

In recent years, the Chinese mitten crab (Eriocheir sinensis), which is widely farmed in China, has been frequently affected by Hepatospora eriocheir. The gut response of mitten crabs to H. eriocheir infection is not yet known. The aim of this study was to evaluate the effects of H. eriocheir infection on the gut microbiota and proteome of E. sinensis. In this study, E. sinensis was divided into two groups based on whether it was infected with H. eriocheir using PCR and Calcofluor white staining. Calcofluor white staining revealed that H. eriocheir was massively aggregated in the gut. Proteomic results showed that 3959 proteins were identified. Under a 1.2-fold change threshold, 420 differentially expressed proteins (DEPs) were screened, including 292 upregulated DEPs and 128 downregulated DEPs. Differential protein expression related to apoptosis and energy metabolism increased, while protein expression related to immune and inflammatory responses and cell motility decreased. In addition, the microbiota results showed that alpha diversity tended to increase, and beta diversity indicated significant differences in gut microbiota between the two groups. Bacteroidetes, Proteobacteria, and Firmicutes were the dominant phyla in the E. sinensis gut. Compared with the control group, Bacteroidetes were significantly reduced in the infected group, while Firmicutes and Tenericutes were significantly increased. At the genus level, the abundance of Bacteroides, Flavobacterium, Shewanella and Aeromonas decreased significantly, while Candidatus Bacilloplasma, Dysgonomonas, Tyzzerella_3 and Lactovum increased significantly. Microbial function prediction found that most differentially abundant bacteria were related to metabolism. At the same time, correlation analysis between the proteome and gut microbiota found potential roles of differential bacteria and proteins involved in metabolism and immunity. These findings suggest that H. eriocheir infection can alter the gut microbiota composition and proteome of E. sinensis.

Cell structure damage contributes to antifungal activity of sodium propylparaben against Trichothecium roseum

Trichothecium roseum is a type of fungus that causes pink rot in muskmelon after the melons are harvested. Pink rot leads to severe decay during storage and causes the production of toxins that can be harmful to human health. Sodium propylparaben (SPP, IUPAC name: sodium; 4-propoxycarbonylphenolate) is an antimicrobial preservative that can be used to treat the inedible parts of fruits in addition to food, medications, and packaging. In this study, the effectiveness of SPP in inhibiting T. roseum was tested, and the inhibition mechanism was investigated. The results show that SPP inhibited the growth and spore germination of T. roseum. The malondialdehyde (MDA) content, propidium iodide staining, alkaline phosphatase (AKP) activity, and calcofluor white (CFW) staining results show that SPP produced a disruption of the cell membrane and cell wall integrity of T. roseum. Scanning and transmission electron microscopy (SEM and TEM, respectively) results also indicate that SPP disrupted the cellular structure of T. roseum. Meanwhile, the large amounts of superoxide anion and hydrogen peroxide in T. roseum accumulated due to the effects of SPP on the activities of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, superoxide dismutase, and decreased catalase. In addition, SPP caused a significant reduction in the incidence rate and disease degree of muskmelon pink rot in vivo. In conclusion, SPP appears to be effective against T. roseum via disruption of the cell membrane and wall. SPP could be used to manage melon pink rot after fruit harvesting because of its disease inhibition effect in vivo.

Clinical and Mycological Characterization of Chronic and Recurrent Dermatophytes using Various Staining and Microscopic Methods

Dermatophytosis is a prevalent infection in tropical and subtropical countries, including India. This study aims to investigate the epidemiology and clinical aspects of chronic and recurrent dermatophytosis, identify the clinical isolates, and assess the effectiveness of different microscopy and staining methods for diagnosis. The objective of the study is to study the epidemiology and clinical aspects of chronic and recurrent dermatophytosis and to identify the clinical isolates. Attempts to improve the diagnostic outcome by implementing different microscopy and staining methods have also been performed. Adult patients with chronic and recurrent cases and positive direct microscopy were included, and clinical details were recorded. The mycological culture was performed. Fifteen isolates were selected randomly and subjected to 0.9% NaCl, Chicago sky blue staining, Calcofluor white staining, and Congo red staining in addition to the standard lactophenol cotton blue (LPCB) preparation. Among the 178 patients in the study, females (56.7%) and patients aged 25-45 (50%) were more frequently affected. Tinea corporis was the most common clinical type (89.32%). Recurrent cases (56.1%) were more prevalent than chronic cases (43.9%). Culture positivity was seen in 60.1%, with Trichophyton mentagrophytes being the most common isolate (86%). Among the staining methods used on 15 selected isolates, Lactophenol Cotton Blue (LPCB) was scored as the most effective, scoring 2.6 out of 3. The study revealed several clinical and epidemiological findings related to dermatophytosis in India, including a high degree of communicability, inadvertent therapies including topical steroid misuse, and a persistent nature requiring an extended duration of therapy. Trichophyton mentagrophytes was the most frequently isolated pathogen. Of the staining methods evaluated, Lactophenol Cotton Blue (LPCB) was found to be the most effective. The findings suggest a need for continued research into effective treatments and diagnostic methods for dermatophytosis in India.

A combinatorial approach of Monarda citriodora essential oil (MEO) and linalool vapors to control fruit rot of Citrus limon caused by a new pathogen, Aspergillus foetidus, and its underlying mode of action.

Citrus limon (lemon) is a widely cultivated citrus fruit. Significant postharvest losses due to fungi plague its production. Environmental and human health hazards have made the application of synthetic fungicides unsuitable. Despite the previous reports of antifungal activities of essential oil (EO) vapors, their synergistic combinations are understudied. Synergistic vapor combinations are advantageous due to less concentration of active components. This study aimed to isolate and identify postharvest fungal pathogens lemon and to evaluate the antifungal effects of synergistic Monarda citriodora EO (MEO)-constituent vapor combinations in vivo and in vitro. Postharvest fungal pathogens of lemon (C. limon) were isolated from various infected samples. The most pathogenic isolate was identified through morphology and its ITS-based rRNA gene sequencing as Aspergillus foetidus (O4). This is the first report of A. foetidus as a postharvest pathogen of lemon. The minimum fungicidal concentrations (MFCs) of MEO vapors treatment against O4 were 1346.15µL/L air. For carvacrol, hexanal, and linalool, MFC was same (96.16µL/L air). Checkerboard assays demonstrated that 1/4 MFC of MEO (336.54µL/L air) and 1/4 MFC of linalool (24.04µL/L air) (M+L) were synergistic against O4. M+L vapors reduced the O4 growth on lemons during storage by 64%±1.50% and preserved their quality (low weight loss %, unchanged pH, increased ascorbic acid content). Propidium iodide staining, ergosterol content analysis, calcofluor white staining and chitin content analysis revealed the integrity loss of the O4 plasma membrane and cell wall. 2',7'-Dichlorofluorescin diacetate staining revealed accumulation of intracellular reactive oxygen species (ROS), and scanning electron microscopy (SEM) analysis exposed the M+L treated mycelia with malformations. M+L vapors offer protection for lemons from A. foetidus and preserve their quality during storage.

621. Evaluation of a Multiplex Endpoint PCR for the Detection of 21 Fungi Commonly Causing Fungal Pneumonia Using Barcoded Magnetic Bead Technology

Abstract Background Fungal pneumonia caused by Aspergillus, Mucorales, and other fungi are potentially life-threatening and often are difficult to diagnose due to similar clinical presentations and poor sensitivity by culture. The BioCode MDx-3000 from Applied BioCode uses Barcoded Magnetic Bead (BMB) technology to digitally detect 21 fungi in a multiplex PCR. We evaluated the BioCode assay using clinically simulated samples and retrospective frozen bronchoalveolar lavage samples. Methods For limit of detection (LOD), 10-20 replicates of each organism were run at 1E4-10 spores/mL. Reproducibility was performed by running three replicates of each target (1E3 spores/mL) within one run, three separate times. Specificity was determined by testing for 37 fungal analytes not targeted by the panel. Matrix specific inhibition was also tested by spiking each organism in the following matrices: whole blood, plasma, serum and BAL. 90 retrospective BAL samples from patients with fungal culture results were run to assess clinical performance. Results LOD of 21 fungal organisms ranging from 19.83 – 271.76 spores/ml. Detection rate was 100% (36/36) in all samples mixed with two fungi; 95% (20/21) in all samples mixed with three fungi. No detection or cross-reactivity was observed in samples containing any of the 37 non-targeted fungi. All replicates were detected in Inter- and intra- reproducibility experiments. No difference or inhibition was observed in detection of targeted fungi spiked in different matrices. Testing retrospective BAL samples showed an overall positive concordance of 61.4% and negative concordance of 98.1%. In 23 BALs positive by both calcofluor white stain (hypha elements) and culture, the BioCode assay correctly detected fungal targets in 78% (18/23) of the samples. Additionally, the assay produced positive detection in 25% of BALs that were culture negative. Limit of Detection and Concordance Limit of detection for the 21 fungal targets. Concordance for each target in retrospective BALs compared to culture. N/A indicates not tested. Conclusion The BioCode assay showed an overall good analytical sensitivity and specificity. Analysis of retrospective BAL samples demonstrated the assay showing good concordance with the culture results. Further studies are warranted to determine the assay’s clinical sensitivity and specificity in aid in diagnosis of fungal pneumonia. Disclosures Kristen Haag, BS, Applied BioCode, Inc.: I am an employee of Applied BioCode|Applied BioCode, Inc.: Stocks/Bonds Michael Aye, PhD, Applied BioCode: Stocks/Bonds Martín Cardona, BS, Applied BioCode, Inc.: I am an employee of Applied BioCode. Sean Zhang, PhD, Applied BioCode: Grant/Research Support|IMMY Diagnostics: Grant/Research Support|KARIUS: Advisor/Consultant|Pearl Diagnostics: Grant/Research Support|Scanogen: Grant/Research Support|T2 Biosystems: Advisor/Consultant|Vela Diagnostics: Grant/Research Support

The antifungal mechanism of Monarda citriodora essential oil, hexanal and their combined vapours on Aspergillus foetidus

BackgroundThe increased reliance on synthetic fungicides for managing postharvest pathogens resulted in the rise of fungicide-resistant pathogen strains. Previous studies have reported essential oils (EOs) as a promising alternative to synthetic fungicides. However, higher concentrations are required for their practical application, which can alter the taste profile of the stored produce. Synergistic combinations of EO volatiles can reduce the active concentrations, thereby mitigating undesirable taste profile changes. However, very few studies have emphasised the changes in the mode of action of EO volatiles when applied as a synergistic combination. This study aimed to decipher the mode of action of Monarda citriodora essential oil (MEO) and hexanal vapour combination (M + H) against the newly identified postharvest pathogen of apple Aspergillus foetidus. ResultsThe M + H vapour treatment synergistically inhibited the spore germination of A. foetidus at the concentration of 144.23 μL L−1 air (MEO) and 96.15 μL L−1 air (hexanal). Propidium Iodide staining and ergosterol content analysis revealed that vapours compromised the plasma membrane integrity. Hence, there was an extracellular leakage of cytoplasmic components, reduced extracellular pH and increased conductivity. Furthermore, calcofluor white staining showed reduced fluorescence intensity, indicating a compromised cell wall. Reduced chitin content in the cell walls of vapour treated mycelia authenticated the results. 2′,7′-Dichlorofluorescin diacetate (DCFDA) staining revealed the generation and accumulation of Reactive Oxygen Species (ROS) within the vapour treated mycelia. ConclusionThe accumulated ROS interferes with ergosterol and chitin synthesis leading to the collapse of the plasma membrane and cell wall; consequently, extracellular leakage occurs.

Microbiological diagnosis of pulmonary invasive aspergillosis in critically ill patients with severe SARS-CoV-2 pneumonia: a bronchoalveolar study

BackgroundDiagnosing COVID-19-associated pulmonary aspergillosis (CAPA) can be challenging since radiological and clinical criteria in the critically ill patient are nonspecific. Microbiological diagnostic support is therefore crucial. The aim of this study was to document the incidence of aspergillosis using bronchoalveolar lavage (BAL) as the diagnostic method and to determine the performance of the current mycological diagnostic tests most widely used for the diagnosis of CAPA, together with evaluation of the Asp lateral flow device (LFD).MethodsProspective cohort study conducted between March 2020 and June 2022. Inclusion criteria were critically ill patients admitted to the ICU with SARS-CoV-2 pneumonia requiring invasive mechanical ventilation. Diagnostic bronchoscopy and BAL were performed at the beginning of invasive mechanical ventilation. The sensitivity, specificity, positive and negative predictive value (PPV and NPV), positive and negative likelihood ratio (LR + and LR-) of BAL culture, direct examination with calcofluor white stain, ELISA (Platelia) and LFD (AspLFD) for detection of galactomannan (GM) were evaluated. Aspergillus-qPCR was applied when discrepancies between diagnostic tests arose.ResultsOf the 244 critically ill patients with SARS-CoV-2 pneumonia admitted to the ICU, the majority (n = 200, 82%) required invasive mechanical ventilation. Diagnostic bronchoscopic procedures were performed in 160 patients (80%), who were enrolled in this study. The incidence of CAPA was 18.7% (n = 30). LFD-GM demonstrated a sensitivity of 84%, specificity of 99%, PPV 94%, NPV 97%, LR(+) of 84, and LR(-) of 0.16. At GM-ELISA indices of ≥ 0.5 and ≥ 1.0, sensitivity was 92% and 79%, specificity was 95% and 99%, PPV 76% and 91%, NPV 99% and 96%, LR(+) 18 and 79, and LR(-) 0.08 and 0.21, respectively. The optimal cut-off index from the ROC curve was 0.48, with sensitivity of 95% and specificity of 95%.ConclusionsUsing a diagnostic strategy based on bronchoscopy and BAL, we documented a high incidence of pulmonary aspergillosis in patients with severe SARS-CoV-2 pneumonia. Asp-LFD showed moderate sensitivity and excellent specificity, with a high PPV, and could be used for rapid diagnosis of patients with suspected CAPA.

A U2 snRNP-specific protein, U2A′, is involved in stress response and drug resistance in Cryptococcus deneoformans

The spliceosome, a large complex containing five conserved small ribonucleoprotein particles (snRNPs) U1, U2, U4, U5 and U6, plays important roles in precursor messenger RNA splicing. However, the function and mechanism of the spliceosomal snRNPs have not been thoroughly studied in the pathogenic yeast Cryptococcus deneoformans. In this study, we identified a U2A′ homologous protein as a component of the cryptococcal U2 snRNP, which was encoded by the LEA1 gene. Using the “suicide” CRISPR-Cas9 tool, we deleted the LEA1 gene in C. deneoformans JEC21 strain and obtained the disruption mutant lea1Δ. The mutant showed a hypersensitivity to 0.03 % sodium dodecyl sulfate, as well as disordered chitin distribution in cell wall observed with Calcofluor White staining, which collectively illustrated the function of U2A′ in maintenance of cell wall integrity. Further examination showed that lea1Δ displayed a decreased tolerance to lower or elevated temperatures, osmotic pressure and oxidative stress. The lea1Δ still exhibited susceptibility to geneticin and 5-flucytosine, and increased resistance to ketoconazole. Even, the mutant had a reduced capsule, and the virulence of lea1Δ in the Galleria mellonella model was decreased. Our results indicate that the U2A′-mediated RNA-processing has a particular role in the processing of gene products involved in response to stresses and virulence.