Fungal Culture Research Articles

Rare case of aspergillosis with solitary renal involvement: micro-fungus ball in graft kidney.

Kidney transplant recipients are at an increased risk of various infections due to immunosuppressive medications. Among them, fungal infections are associated with high mortality and morbidity. This report presents the case of a 54-year-old kidney-transplant recipient who was diagnosed with aspergillosis with solitary renal involvement. He was diagnosed by kidney biopsy with the micro-fungus ball. In the biopsy sample, consisting mostly of the medulla, a small focus consisting of an aggregate of fungal microorganisms was identified. The micro-fungus ball, which was also present in serial sections, was characterized by slight pigmentation and septate hyphae with acute angle branching, highlighted by the silver stains. The patient was examined for invasive fungal infection. In CT scans, there were no signs of invasive fungal infection. Due to the unexpected kidney biopsy finding, the patient underwent a repeat allograft biopsy from which a culture was sent. Aspergillus fumigatus complex was detected in tissue fungal culture of this repeat biopsy. The patient was started on voriconazole treatment and was successfully treated. It should be kept in mind that fungal infections with isolated subtle renal involvement may be possible in KTR under immunosuppressive treatment without an obvious fungal focus being demonstrated by imaging methods.

Morphotype Analysis of Endophytic Fungus Isolated from Taxus sumatrana of Mount Singgalang, West Sumatra Region

Taxus sp. plants in temperate climates like Europe and America have been extensively studied and identified as promising sources of endophytic microbes that produce anticancer compounds such as Taxol. These endophytic microbes exhibit high adaptability to the specific growing conditions of their host plants. Research has highlighted metabolic differences between tropical and temperate endophytes. The presence of Taxus sumatrana plants in tropical areas like Mount Singgalang in West Sumatra, Indonesia was subjected to explore potential new sources of bioactive compounds from tropical microbes. This research aims to identify the morphotypes of endophytic fungi isolated from the barks of Taxus sumatrana plants originating from Mount Singgalang. The method used is small pieces of the inner bark of Taxus sumatrana were placed on the surface of growth medium (PDA). The small hyphae emerging from the piece of plant samples were regularly monitored for the morphological characteristics of the colony and conidia structure. Each fungal culture was checked for purity and transferred to another PDA plate by the hyphal tip method. The investigation isolated two endophytic fungi from the bark of Taxus sumatrana: Pestalotiopsis microspora and Neopestalotiopsis sp.

Induction of Three New Secondary Metabolites by the Co-Culture of Endophytic Fungi Phomopsis asparagi DHS-48 and Phomopsis sp. DHS-11 Isolated from the Chinese Mangrove Plant Rhizophora mangle.

Co-cultivation is a powerful emerging tool for awakening biosynthetic gene clusters (BGCs) that remain transcriptionally silent under artificial culture conditions. It has recently been used increasingly extensively to study natural interactions and discover new bioactive metabolites. As a part of our project aiming at the discovery of structurally novel and biologically active natural products from mangrove endophytic fungi, an established co-culture of a strain of Phomopsis asparagi DHS-48 with another Phomopsis genus fungus DHS-11, both endophytes in mangrove Rhizophora mangle, proved to be very efficient to induce the production of new metabolites as well as to increase the yields of respective target metabolites. A detailed chemical investigation of the minor metabolites produced by the co-culture of these two titled fungal strains led to the isolation of six alkaloids (1-6), two sterols (7, 8), and six polyketides (9-14). In addition, all the compounds except 8 and 10, as well as three new metabolites phomopyrazine (1), phomosterol C (7), and phomopyrone E (9), were not present in discrete fungal cultures and only detected in the co-cultures. The structures were elucidated on the basis of spectroscopic analysis, and the absolute configurations were assumed by electronic circular dichroism (ECD) calculations. Subsequently, the cytotoxic, immunosuppressive, and acetylcholinesterase inhibitory properties of all the isolated metabolites were determined in vitro. Compound 8 exhibited moderate inhibitory activity against ConA-induced T and LPS-induced B murine splenic lymphocytes, with IC50 values of 35.75 ± 1.09 and 47.65 ± 1.21 µM, respectively.

Sporothrix brasiliensis-specific polymerase chain reaction for the diagnosis of cat and human sporotrichosis through non-invasive samples.

Zoonotic sporotrichosis caused by Sporothrix brasiliensis is an emerging mycosis in Latin America. One of the problems to quickly treat infected animals and break the transmission chain is associated with the time-consuming gold-standard diagnosis method (culture). We aimed to evaluate a species-specific polymerase chain reaction (PCR) for the diagnosis of sporotrichosis caused by S. brasiliensis using non-invasive samples. We performed a retrospective cross-sectional study using samples collected with swabs from humans and cats with clinical suspicion of sporotrichosis. Deoxyribonucleic acid (DNA) was extracted using a commercial kit, and a species-specific PCR for S. brasiliensis detection was performed. One hundred ten samples were included. PCR showed a good concordance with culture (86% of agreement) for human and cat samples (Kappa coefficient=0.722, and 0.727, respectively). In conclusion, our data shows that this adapted PCR using non-invasive samples can be applied to sporotrichosis diagnosis, being a good alternative mainly in regions with a lack of mycologists to identify the fungus in culture, contributing to the control of this emergent zoonosis.

First report of Colletotrichum scovillei causing anthracnose fruit rot of pepper in Ohio.

In August 2019, the Ohio State University Vegetable Pathology laboratory received multiple bell and banana pepper fruits (Capsicum annuum, cvs. unknown) from Columbiana County, Ohio. The grower reported a disease incidence of 100% and severity of 70% in fruits across their pepper fields. Fruit lesions were brown, sunken, and covered with orange-colored sporulation. On banana peppers, the lesions mainly affected the blossom end of the fruits, while the lesions were distributed randomly on bell pepper fruits. Pieces of diseased tissue were cut from the fruit and surface sterilized in 0.5-0.6 % sodium hypochlorite, rinsed in sterile water, blotted dry, and placed on potato dextrose agar. All of the fungal cultures recovered were cottony, pale gray-green with shades of orange on the underside of the mycelial mat. Two representative isolates, SM209-19 (bell pepper) and SM210-19 (banana pepper), were grown on oatmeal agar to induce sporulation. Pink-orange concentric rings containing acervuli and conidia were present on the oatmeal agar plates after one week of growth at 22◦C (12-h dark/light). Conidia (n=29) were hyaline, aseptate, cylindrical in shape, and had an average length of 10.5 µm (std. dev. = 1.3 µm) and width of 4.1 µm (std. dev. = 0.6 µm) (Fig.1). DNA was extracted from both isolates using a DNeasy Plant Kit (Qiagen Inc, Germantown, MD), and partial sequences of the internal transcribed spacer (ITS) region, -tubulin 2 gene (TUB2), and glyceraldehyde-3-phosphate dehydrogenase gene (GDPH) were amplified by PCR with the following primers: ITS4/ITS5 (White et al. 1990), Bt1a/Bt1b (Glass et al. 1995), and GDF1/GDR1 (Guerber et al. 2003), and squenced. The ITS region of both isolates SM209-19 and SM210-19 (PP280815 and PP280816, respectively) showed 100% identity with C. scovillei (Cs) isolate LJTJ35 (KP748226). The partial sequences of GDPH, (PP320348, PP320349, respectively) showed 99% sequence identity with the Cs CBS 126528 (JQ948597) and 100% identity with Cs HP1 (MT826948) The partial sequences of TUB2 (PP472464 and PP472465, respectively) had 100% sequence similarity with the Cs HP1 and Cs CBS 126528 (MT826951, JQ949918 respectively). Pathogenicity was tested in a greenhouse experiment on blossoming bell pepper plants (cv. Carmen) by spraying 10 ml of 1 X 105 conidia/ml suspension onto flower blooms (nine plants per isolate). Control pepper plants were mock inoculated by spraying 10 mL of sterile deionized water. The plants were re-inoculated one week later. Brown, sunken lesions with orange sporulation developed on the fruits of inoculated plants 21 days after the initial inoculation (Fig. 2), while the mock-inoculated plants did not produce any symptoms. Culturing from symptomatic fruits on PDA, following the same method described above, produced fungal colonies with the same morphological traits previously described. C. scovillei causing anthracnose on pepper has been described in the US (Toporek et al. 2021), Brazil (Caires et al. 2014), China (Zhao et al. 2016), and different South Asia Countries (Khalimi et al. 2019). Open-field peppers are produced in Ohio on more than 5,400 acres, with a value of more than $53 million, with anthracnose being one of the most severe fungal diseases reducing yield. This newly reported Colletotichum species could represent a further threat for this crop. Further studies evaluating fungicide sensitivity and efficacy against this pathogen will be of crucial importance for disease management.

Exploring the biotechnological prospects of a recently discovered fungus isolated from marine mucilage

AbstractThis study aimed to determine the usability of the identified fungus isolated from the marine-mucilage formation in the biotechnological processes. For this purpose, the antimicrobial and antioxidant activities of the extract obtained from the fungus were examined, and the fatty acid methyl ester composition was determined. The molecular identification of the pure fungal culture was made using LSU regions. In addition, fungal extracts were prepared using different solvents, and the antimicrobial activity of these extracts was investigated by disk diffusion and minimum inhibition concentration methods. At the same time, the antioxidant properties of these extracts were analyzed using the DPPH and ABTS free radical removal methods. Also, FAME analysis was performed to determine the fatty acid content of the fungal extract. According to the study results, the new isolate was identified as the fungus Rhizopus stolonifer. Although fungal extracts have no significant antimicrobial activity, it has been determined that they performed successful DPPH and ABTS scavenging activity without needing additional reactions. FAME results indicate that the mucilage-originated fungus R. stolonifer is a valuable provider of fatty acids that, when purified at large bioreactors, can be good and cheap sources of next-generation biologicals for wide-ranging biotechnological applications.

Contemporary Update on the Microbiology of Paranasal Sinusitis

Background: Sinusitis, whether acute or chronic, is likely due at least in part to disruptions in the microbiota of the paranasal sinuses. Sinus cultures are often employed to guide medical treatment. Objective: To quantify the contemporary microbiology of the paranasal sinuses and better understand the utility of paranasal sinus cultures. Methods: We identified patients from 2018 to 2019 with sinus cultures taken by an otolaryngologist in the outpatient setting in our healthcare system with a concurrent diagnosis of acute or chronic rhinosinusitis. These cultures were analyzed based on their culture type and result. The most commonly isolated bacteria were further analyzed by species; Staphylococcus resistance patterns were analyzed as well. Results: A total of 2302 culture samples were collected: 2012 (87%) bacterial, 287 (13%) fungal, and 3 (0.1%) mycobacterial cultures. The results of more than half (1142, 57%) of these bacterial cultures were positive for a named genus, while those of 592 (29%) were positive for normal sinus flora and 16 (0.8%) for normal oral flora, and those of 183 (9%) showed no growth. The results of another 79 (4%) bacterial cultures were positive for unnamed bacteria, which were not further classified (e.g., Gram-negative rods). Of the positive bacterial cultures with named genera, the most common genera identified was Staphylococcus (383, 34%). Of these, the most common species of Staphylococcus was S. aureus (311, 81%), 42 of which (14%) showed methicillin resistance (MRSA). Of the fungal cultures, 265 (92%) resulted in no growth, and all three mycobacterial cultures showed no growth. Conclusions: In contrast to fungal cultures, the majority (57%) of sinus bacterial cultures showed positive results, with the identification of a named genus, highlighting the potential utility of this assay in guiding medical therapy.

Lecanicillium psalliotae (Hypocreales: Cordycipitaceae) Exerts Ovicidal and Larvicidal Effects against the Sheep Blood-Feeding Nematode Haemonchus contortus through Its Liquid Culture Filtrates.

Nematophagous fungi (NF) form part of the soil microbiota and are natural enemies of nematodes, helping to regulate nematode populations. A verticillate NF isolated from soil from Tepalcingo, Mexico, was morphologically and molecularly characterised. This fungus was cultured in two different liquid media-Czapek-Dox broth (CzDoxB) and sweet potato dextrose broth (SPDB)-for 21 days. The ovicidal (OA) and larvicidal (LA) activities of fungal liquid culture filtrates (LCFs) were assessed in 96-well microtitre plates at different concentrations against Haemonchus contortus after 48 h. The morphological and molecular identification revealed the presence of Lecanicillium psalliotae. Additionally, the groups of compounds associated with nematocidal activity were determined from a qualitative chemical profile (QCP) using different reagents. The highest OA of the LCFs was obtained at 25 mg/mL from SPDB and CzDoxB and amounted to 97.2 and 99.06%, respectively. Meanwhile, the highest LA recorded with these LCFs at 100 mg/mL was 54.27% and 96.8%, respectively. The QCP revealed the presence of alkaloids and tannins in both LCFs that have previously been associated with nematocidal activity. Lecanicillium psalliotae exerted an important effect on H. contortus and could be of significance in future studies focused on the control and prevention of haemonchosis in small ruminants.

Novel foodborne source of Xerochrysium xerophilum (Pitt) Pitt, raw peanuts stored at low moisture and high CO2

Peanut seed is ideally stored at low water activity (aw) and low temperature, since common mycotoxigenic fungi cannot survive below 0.8 aw, and the life cycle of seed-infesting insects is interrupted below 15 °C. Hermetic storage bags at 0.77 aw, room temperature, and ≥ 9000 mg/m3 CO2, were tested for their potential use in countries where temperature control is unaffordable. Despite these inhospitable conditions, peanuts displayed an unusual white powder that we identified as the xerophilic fungus Xerochrysium xerophilum (syn. Chrysosporium xerophilum), and sequenced its genome. Previously, only 19 sequences shorter than 1700 nt were available. For six decades, only three food sources of X. xerophilum were known: dry prunes, chocolate, and dry coconut. Here we report peanut as a novel food source of this extremophile and make one isolate available at the USDA culture collection. Given the slow growth of this fungus in culture, we developed a robust PCR to sequence and identify X. xerophilum on contaminated peanut seed without performing DNA extraction. The broad range of oxygen, aw and temperatures that support growth of X. xerophilum will significantly affect the decisions made regarding the conditions to safely store peanuts.

Advances in plastic mycoremediation: Focus on the isoenzymes of the lignin degradation complex

This study investigates P. ostreatus and A. bisporus biodegradation capacity of low density polyethylene (LDPE) oxidised to simulate environmental weathering. Fourier transform infrared (FT-IR) spectroscopy and scanning electron microscopy (SEM) were used to analyse the degradation of LDPE treated with fungal cultures. Molecular implications of LDPE degradation by P. ostreatus and A. bisporus were evaluated by Reverse transcription followed by quantitative PCR (qRT-PCR) of lac, mnp and lip genes expression. After 90 days of incubation, FT-IR analysis showed, for both fungal treatments, an increasing in the intensity of peaks related to the asymmetric C-C-O stretching (1160 to 1000 cm−1) and the -OH stretching (3700 to 3200 cm−1) due to the formation of alcohols and carboxylic acid, indicating depolymerisation of LDPE. This was confirmed by the SEM analysis, where a widespread alteration of the surface morphology was observed for treated LDPE fragments. Results revealed that the exposure of P. ostreatus to oxidised LDPE treatment led to a significant increase in the expression of the lac6, lac7, lac9, lac10 and mnp2 genes, while A. bisporus showed an over-expression in lac2 and lac12 genes. The obtained results offer new perspectives for a biotechnological use of P. ostreatus and A. bisporus for plastic bioremediation.

Nano Zinc Oxide Particle Synthesis from Bio-Waste Selaginella willdenowii Leaf Extract: A Multi-Faceted Approach for Environmental and Biomedical Applications

Selaginella willdenowii, a commonly used greenhouse fern, was often used as a biowaste to synthesize zinc oxide nanoparticles (ZnO NPs) in an eco-friendly and cost-effective way. UV–Visible spectra studies were carried out to confirm the synthesis of S. willdenowii-mediated ZnO NPs (SW-ZnO NPs), and a peak at 367[Formula: see text]nm with a sharp band gap of 3.415[Formula: see text]eV was observed. The X-ray diffraction analysis indicated that the crystalline size of the synthesized SW-ZnO NPs was 11.971[Formula: see text]nm. The phytochemicals present in the extracts and the compounds involved in the reduction of metal to nanoparticles were determined by Fourier Transform Infrared analysis. Scanning electron microscopy was utilized to analyze the surface morphology and size of the obtained SW-ZnO NPs. The examination revealed that they exhibited a hexagonal shape, with an average size falling within the range of 17–23[Formula: see text]nm. Under ultra-violet light, reactive blue 220 and reactive yellow 145 dyes showed 78.06% and 60.14% degradation, showing potential photocatalytic degradation activity. The synthesized SW-ZnO NPs also exhibited antimicrobial activity against bacterial strains (Escherichia coli and Bacillus subtilis) and fungal cultures (Candida tropicalis and Candida albicans) showed cytotoxic activity against Hep-G2 cell lines. Our results suggest the green synthesized SW-ZnO NPs have potential photocatalytic, antimicrobial and cytotoxic potential.

Case Report : Navigating the uncommon: Medicopsis romeroi-induced corneal ulcer

Abstract* This report details a rare case of corneal infection caused by Medicopsis romeroi, highlighting clinical presentation, diagnostic methods, treatment, and outcome. An early adolescence male, after recent corneal tear repair, presented acute eye pain, redness, and decreased vision for 1 day. He underwent corneal tear repair elsewhere post vegetative matter-related swimming injury. Ophthalmic examination revealed a 2.5*3.3mm ulcer around sutures. Corneal scrapings enabled microscopy and culture. With lesion worsening, tissue adhesive (TA) and bandage contact lens (BCL) were applied. Fungal culture identified Medicopsis romeroi. Patient's history, clinical course, and treatment were documented. Microscopy unveiled fungal hyphae, later confirmed as Medicopsis romeroi. Treatment included topical/systemic antifungals. Post-TA + BCL, significant improvement followed, with re-epithelialization, scarring. This report underscores the importance of considering rare fungal pathogens like Medicopsis romeroi in cases of corneal ulcers, especially in individuals with unusual risk factors. Timely diagnosis through microscopy and fungal culture, coupled with tailored antifungal treatment led to successful management and favorable patient outcomes. This case also emphasizes the significance of vigilant clinical observation, precise laboratory analysis, and multidisciplinary collaboration between ophthalmologists and microbiologists in managing atypical ocular infections.

Mortierella Ramanniana Lipid Fermentation Wastewater as an Innovative Maceration Liquid Medium for Sustainable Solid-State Cultivation of Higher Fungi

Mortierella Ramanniana Lipid Fermentation Wastewater as an Innovative Maceration Liquid Medium for Sustainable Solid-State Cultivation of Higher Fungi

Endophytic Aspergillus fumigatiaffinis: Novel paclitaxel production and optimization insights

This study investigated the potential of endophytic fungi to produce paclitaxel (Taxol®), a potent anticancer compound widely employed in chemotherapy. This research aimed to identify, confirm, and characterize endophytic fungi capable of paclitaxel (PTX) production and assess their paclitaxel yield. Additionally, it aimed to investigate factors influencing paclitaxel production. A total of 100 endophytic fungal isolates were collected and identified from the roots of Artemisia judaica. Aspergillus fumigatiaffinis exhibited the highest PTX production (26.373 μg L−1) among the isolated endophytic fungi. The strain was identified as A. fumigatiaffinis (Accession No. PP235788.1). Molecular identification confirmed its novelty, representing the first report of PTX production by A. fumigatiaffinis, an endophyte of Artemisia judaica. Optimization through full factorial design of experiments (DOE) and response surface methodology (RSM) significantly enhanced PTX production to 110.23 μg L−1 from 1 g of dry weight of the fungal culture under optimal conditions of pH 8.0, 150 μg L−1 becozyme supplementation, and 18 days of fermentation in potato dextrose broth. The presence of paclitaxel was confirmed using thin layer chromatography, high performance liquid chromatography, and gas chromatography–mass spectrometry. These findings maximize the role of endophytic fungus to produce a secondary metabolite that might be able to replace the chemically produced PTX and gives an opportunity to provide a sustainable source of PTX eco-friendly at high concentrations.Key points• Endophytic fungi, like A. fumigatiaffinis, show promise for eco-friendly paclitaxel production• Optimization strategies boost paclitaxel yield significantly, reaching 110.23 μg L−1• Molecular identification confirms novelty, offering a sustainable PTX source

Clinical Analysis of Patients Diagnosed with Cutaneous Sporotrichosis in China.

This study aimed to improve the understanding of sporotrichosis by analyzing the epidemiological characteristics, clinical manifestations, mycological features, and pathological characteristics of the disease in eastern China. Clinical data of 49 patients diagnosed with cutaneous sporotrichosis in dermatology clinics over a 20-year period were collected and analyzed retrospectively. The analysis included patient demographics, occupations, clinical types, lesion sites, misdiagnosis rates, laboratory investigations, treatment and outcomes. The study included 22 male and 27 female patients, with a mean age of 52.4 years. Farmers (42.86%) and manual workers (28.57%) had a higher risk of infection. The most common clinical types were lymphocutaneous (30.61%) and fixed (69.39%), predominantly affecting the face and upper limbs. Misdiagnosis as other infectious skin diseases occurred in 35 patients (71.43%). Fungal culture and histopathological examination were important diagnostic tools. Treatment with oral itraconazole for three months led to relief and regression of the skin lesions in most patients, although a few experienced recurrences. Cutaneous sporotrichosis mainly affects individuals working in agriculture and manual labour, with lymphocutaneous and fixed types being the predominant clinical manifestations. The high misdiagnosis rate emphasizes the importance of early recognition, accurate diagnosis and standardized treatment for the prognosis and cure of sporotrichosis. Fungal culture and histopathological examination are essential for diagnosis, and oral itraconazole is an effective treatment option.

ICU care through NGS – Based identification of infectious agents: A comparative study

BackgroundSepsis claims 1 in 5 lives annually as per global statistics. Sepsis incidence in recent studies represents at least 35 % of all ICU admissions and has a high mortality rate, especially in the presence of co-existing morbidities. The challenge has been to accurately diagnose the causative organism, considering factors such as possible polymicrobial infections, commensals and environmental contaminants. Legacy techniques such as culture, automated culture systems or even newer species-specific PCR or film array these challenges difficult to overcome. The Bactfast® and Fungifast® assays along with the integrated workflow is based on next generation sequencing and have the ability to demarcate infecting pathogen from contamination and commensal. The unique ability to pinpoint the exact pathogen, considering the commensal and contamination in a variety of samples, with an extremely high sensitivity could lead it to be a tool of diagnostic choice for non-resolving ICU sepsis due to its comprehensive coverage and speed. The aim of this study was to evaluate the use of Bactfast® and Fungifast® as a last mile diagnostic tool in a ICU setting. MethodThis study was carried out considering access to four intensive care units (ICU). Legacy testing, mostly done on culture, was conducted at the various integrated microbiology facilities of the hospitals where the ICUs were located, in Chennai, India. NABL accredited laboratory Micro Genomics (India) Pvt Ltd, was established as the central processing facility for next generation sequencing to run the Bactfast® and Fungifast® assay. Co-relation of results for 490 samples was done retrospectively by a multi-disciplinary team of consultants which comprised of microbiologists, and infectious disease physicians. ResultThe diagnostic workflow established with the Bactfast® assay provided a sensitivity of 94.1 % and specificity of 86.6 %. Identification of pathogens in Bactfast® was better when compared to the data published in 2017, as reflected by positive co-relation with clinical confirmation. Although the Fungifast® specificity was high, at 99.4 %, only 12 samples were positive on fungal culture out of 490 samples. Therefore, it was concluded a further study for fungi based on multiple technologies with more true positive samples is required to evaluate the test. ConclusionBactfast® can identify pathogens in a sample without any bias. Its introduction as diagnostic modality in life threatening ICU sepsis could reduce mortality and morbidity. Although the initial results of Fungifast® are encouraging a further research is required for more information on test sensitivity.

A Rapid PCR-Based Diagnostic Method for Skin Infection with Mycobacterium marinum.

The increasing incidence of chronic skin infections caused by Mycobacterium marinum, coupled with the time-consuming and low detection rates nature of traditional culture and histological-based diagnostic methods, underscores the need for an expedited approach. The study aims to develop a rapid and efficient method for detecting M. marinum with PCR technology. We designed four pairs of primers based on DNA sequences from GeneBank and prior studies, we utilized both PCR and Real-time PCR to identify M. marinum. Specificity and sensitivity assessments were conducted in vitro by DNAs extracted from M. marinum and other bacterial or fungal cultures. Further validation was performed through the implementation of a mouse skin infection model to optimize and confirm the efficacy of the detection method in both fresh and paraffin-embedded skin tissues. The same PCR testing system was further confirmed with paraffin-embedded skin tissues samples from patients as well. The results of the study indicate promising outcomes for the four-pair primers system. It demonstrated 100% sensitivity in detecting M. marinum from purified cultures, including typical strains and nine clinical isolates, while achieving a specificity of 100%. This specificity was evidenced by the absence of PCR products from 12 bacterial species, 12 fungi species, and six other non-tuberculous mycobacterium (NTM) species. In the animal model, the PCR assay exhibited high detection efficacy for both infected fresh tissues and paraffin-embedded tissues, with a slight superiority observed in fresh tissues. However, the PCR assay exhibited high detection efficacy for clinical paraffin-embedded tissues. These findings collectively underscore the robust detection capabilities of our four-pair primers in both in vitro and in vivo settings. A sensitive and highly specific rapid detection system has been successfully developed that can be used to detect M. marinum in both infected fresh tissues and paraffin-embedded tissues.

Fungal cryopreservation across 61 genera: Practical application and method evaluation

ABSTRACT Fungi occupy important environmental, cultural, and socioeconomic roles. However, biological research of this diverse kingdom has lagged behind that of other phylogenetic groups. This is partially the result of the notorious difficulty in culturing a diverse array of filamentous fungal species due to their (i) often unpredictable growth, (ii) unknown preferences for culturing conditions, and (iii) long incubation times compared with other microorganisms such as bacteria and yeasts. Given the complexity associated with concurrently culturing diverse fungal species, developing practical methods for preserving as many species as possible for future research is vital. The widely accepted best practice for preserving fungal tissue is the use of cryogenic biobanking at −165 C, allowing for the preservation and documentation of stable genetic lineages, thus enabling long-term diversity-centered research. Despite the extensive literature on fungal cryopreservation, substantial barriers remain for implementation of cryogenic biobanks in smaller mycological laboratories. In this work, we present practical considerations for the establishment of a fungal culture biobank, as well as provide evidence for the viability of 61 fungal genera in cryogenic storage. By providing a pragmatic methodology for cryogenically preserving and managing many filamentous fungi, we show that creating a biobank can be economical for independently owned and operated mycology laboratories, which can serve as a long-term resource for biodiversity, conservation, and strain maintenance.

Evaluation of Two Readings for the QuicGM Aspergillus Lateral Flow Assay in a Group of Immunocompromised Patients in Iraq

Background: Aspergillus spp. causes a wide range of diseases called Invasive Aspergillosis (IA) which is a fatal illness that affects a variety of immunocompromised people worldwide. Objective: Using the lateral flow test for the detection of Galactomannan antigen in immunocompromised patients suspected to have IA. Patients and Methods: This study was conducted on 72 patients, whose samples (serum, Bronchoalveolar lavage, Blood) were collected from the Hematology-Oncology Unit at Baghdad Teaching Hospital and Pediatric Welfare Hospital, and ICU in Ghazi AL-Hariri Surgical Specialties Hospital, and Bone Marrow Transplantation Center in period between November 2022 to February 2023. Patients’ blood and sputum were sent for fungal culture to confirm the diagnosis in most cases. Results: QuicGM Aspergillus Lateral Flow Assay was conducted on 72 patients, of whom 34 () were positive and 38 (52.7) were negative (P-value of 0.001 and 0.5) respectively. This screening aimed at detecting IA. One week later, the second confirmative result was obtained from 24 patients to determine the response to antifungal drugs or recovery from neutropenia on which 15 readings were negative and nine were positive. Out of 48 single readings, 25 were positive and 23 were negative. All for 72 members of the control group gave negative results. This study is the first in Iraq to use this assay. Conclusion: QuicGM Aspergillus Lateral Flow Assay was found to be reliable, sensitive, and specific, and proved to be a very good guide for the early diagnosis of IA in immunocompromised patients and in monitoring treatment outcomes and follow-up.

Reliability of the chemical, metabolic, and molecular methods in discriminating aflatoxigenic from non-aflatoxigenic Aspergillus isolates

Aflatoxins, potent carcinogens produced by Aspergillus species (A. flavus and A. parasiticus), are primarily consumed through dietary sources. Aflatoxin management using non-aflatoxigenic Aspergillus to outcompete their aflatoxigenic congeneric to achieve low residual toxin levels is a promising biological control (BC) field intervention. A prerequisite for the BC program is the accurate delineation of aflatoxigenic fungi from their non-aflatoxigenic relatives. In this study, we use three methods: Chemical (Dichlorvos-ammonia), Metabolic (ELISA coupled live cultures), and Molecular (10 aflatoxin biosynthetic genes) to determine the aflatoxigenicity of Aspergillus spp. previously collected from Eastern Kenya, an aflatoxin hotspot. Thirty-six different Aspergillus isolates were identified and studied microscopically from a total of 314 fungal cultures. The chemical method reported 21 aflatoxigenic and 15 non-aflatoxigenic isolates, while the metabolic technique distinguished 20 aflatoxigenic and 16 non-aflatoxigenic isolates. A large proportion (97.22 %) of the isolates harboured the aflR, aflO, and aflM genes, while 91.67 % of the isolates possessed the aflS gene. All isolates (100 %) harboured the aflD, aflQ, aflP, and aflK genes. ELISA-coupled live cultures positively correlated with Dichlorvos-ammonia (p > 0.01), only differing in one sample, indicating a strong concurrence between the two methods at segregating toxigenic A. flavus isolates. Cluster analysis based on Multiple Correspondence revealed that the non-aflatoxigenic group exhibited similar characteristics correlating with the absence of aflR, aflS, aflI, and aflO genes. These findings indicate the reliability of the Chemical and Metabolic methods and can function as confirmatory assays to complement the molecular detection approach in determining aflatoxigenicity among Aspergillus spp.