Multiple Aspergillus Research Articles

Identification of Peanut Kernels Infected with Multiple Aspergillus flavus Fungi Using Line-Scan Raman Hyperspectral Imaging

Identification of Peanut Kernels Infected with Multiple Aspergillus flavus Fungi Using Line-Scan Raman Hyperspectral Imaging

S2.3c High-resolution digital DNA melting: a breakthrough in diagnosing IMI?

S2.3 Novel diagnostic tools for invasive mold infection, September 21, 2022, 3:00 PM - 4:30 PMInvasive mold infections (IMIs) such as Aspergillosis, Mucormycosis, Fusariosis, and Lomentosporiosis have emerged as important pathogens in immunocompromised patients, with mortality rates as high as 50% to nearly 80% for these infections. Outcomes can be substantially improved with early initiation of appropriate antifungal therapy, yet early diagnosis remains difficult to establish and often requires multidisciplinary teams evaluating clinical and radiological findings plus supportive microbiologic findings. Conventional fungal culture and PCR techniques are limited by low sensitivity, long turn-around time, and are insufficient for differentiating between infection and colonization. Other non-culture-based tests, such as the Galactomannan test, are available only for invasive aspergillosis and are limited by imperfect test performance. Better and more rapid diagnostics are needed to enable early diagnosis and targeted treatment of IMIs and improve survival. We have developed a new technology called digital high resolution melting analysis (dHRM) to enable a rapid and robust diagnosis of IMI. This technology accomplishes fast genotyping of fungal genomic sequences in clinical samples by: (1) conducting broad-based amplification of fungal genes in a digital PCR format, and (2) conducting high-resolution melting of the DNA amplicons in each digital reaction. High resolution melting measures the fluorescence of a saturating intercalating dye as dPCR-amplified fungal DNA fragments are rapidly heated and disassociated, producing sequence-defined melt curves in a closed reaction format. These melt curves serve as unique fungal ‘fingerprints’, which allows us to correctly identify disease-causing pathogen(s) with an accuracy of 99%-100% through the use of machine learning methods trained to tolerate variations in reaction conditions. This approach provides a simple, low-cost, fast, and robust method for pathogen identification. Here, we will present the performance of this new technology on clinical bronchoalveolar lavage (BAL) samples from patients with and without IMI. A total of 75 patient BAL samples were tested, coming from 30 patients with proven (n = 1), probable (n = 25), or putative (n = 4) invasive pulmonary aspergillosis (IPA) infections, and from 45 patients classified as not having IPA (n = 4 possible IPA and n = 41 no IPA). Our results show that dHRM was able to reliably differentiate between different Aspergillus spp. and was also able to identify Aspergillus spp. in BAL samples from patients with probable IPA where qPCR had resulted in negative. In a few instances, it also detected rare molds in culture-negative samples and multiple Aspergillus spp. within one BAL sample, a phenomenon that has been previously described in cultures. Interestingly, there was no clear correlation between dHRM results and BAL galactomannan levels, suggesting that dHRM may serve as an independent method allowing for a combined diagnostic approach. While further validation and optimization are needed, these results suggest that dHRM is a promising new diagnostic technology capable of providing clinically relevant information within 2-3 h.

P154 Fungal spore count in a tertiary care hospital in Mumbai, India

Poster session 2, September 22, 2022, 12:30 PM - 1:30 PM ObjectivesFungal spores are ubiquitously present in the environment. Appropriate temperature and humidity can lead to fungal growth. It is of concern in a hospital environment. Diabetic and immunosuppressed persons are more vulnerable to such infections. The fairly humid and slightly warm climate of Mumbai is ideal for fungal growth. According to an article released in March 2021, the total fungal count in Mumbai was 16.2 CFUs. Here is a brief gist of how fungal spores were controlled within a tertiary hospital in Mumbai, India which is located near sea by simple but effective methods.MethodsAir sampling of the Operation Theatre (OT), Central Sterile Supply Department (CSSD), Bone marrow transplant (BMT) areas is done on weekly basis in the hospital and for areas such as ICU (Intensive Care Unit), Oncology (ONCO) Ward, Cardiac Catheterization (Cath) lab and Digital subtraction angiography (DSA) lab monthly air sampling is done to check for bacterial and fungal load. Two types of air samplers are used: Heico air sampler from HiMedia and Dyanamicro air sampler. OT, BMT, CSSD have HEPA (high efficiency particulate air) filters installed for filtration of air. The other areas like ICU, ONCO ward, Cath lab, and DSA lab do not have HEPA filters.ResultsIn the last 4 years there were two instances where fungal growth was observed once in BMT and once in ICU. In ICU Aspergillus niger growth was observed and in BMT there was a growth of multiple Aspergillus spp. It took nearly 3-4 months for the eradication of the fungus in BMT and 2 months to clear the fungus from ICU. Due to strict engineering control, we were able to restrict the spread within hospital.In BMT unit the growth was observed during rainy season when because of heavy rain there was seepage in one wall of the unit and also there was a leakage of sink in the ante-room area. When these repairs were done the fungal growth was not observed afterward.In ICU thorough cleaning of the Air Handling Unit (AHU) filter leads to resolution of the issue.Both these episodes were uneventful as no patient had any infection due to these.Factors that helped in controlling the fungal spores spread are:Controlled temperatureHumidityAC duct cleaningRegular AHU filter cleaningDifferent sizes of filters used for filtration of circulating air in hospitalNumber of air changesUse of antifungal paints in the hospitalRegular checks in hospital for any leakage or seepage issues.Monitoring of constructional activities in the hospital with appropriate measures to control fungal spore disseminationConclusionEven though the hospital is situated near sea and the outside air has high humidity and high microbial load, effective engineering measures and close monitoring of the areas can help in controlling the spread of fungus within the hospital. Routine periodic checks of the air for fungal spores can also help prevent nosocomial infections.

Aspergillus Hydrophobins: Physicochemical Properties, Biochemical Properties, and Functions in Solid Polymer Degradation.

Hydrophobins are small amphipathic proteins conserved in filamentous fungi. In this review, the properties and functions of Aspergillus hydrophobins are comprehensively discussed on the basis of recent findings. Multiple Aspergillus hydrophobins have been identified and categorized in conventional class I and two non-conventional classes. Some Aspergillus hydrophobins can be purified in a water phase without organic solvents. Class I hydrophobins of Aspergilli self-assemble to form amphipathic membranes. At the air–liquid interface, RolA of Aspergillus oryzae self-assembles via four stages, and its self-assembled films consist of two layers, a rodlet membrane facing air and rod-like structures facing liquid. The self-assembly depends mainly on hydrophobin conformation and solution pH. Cys4–Cys5 and Cys7–Cys8 loops, disulfide bonds, and conserved Cys residues of RodA-like hydrophobins are necessary for self-assembly at the interface and for adsorption to solid surfaces. AfRodA helps Aspergillus fumigatus to evade recognition by the host immune system. RodA-like hydrophobins recruit cutinases to promote the hydrolysis of aliphatic polyesters. This mechanism appears to be conserved in Aspergillus and other filamentous fungi, and may be beneficial for their growth. Aspergilli produce various small secreted proteins (SSPs) including hydrophobins, hydrophobic surface–binding proteins, and effector proteins. Aspergilli may use a wide variety of SSPs to decompose solid polymers.

CYP51 Mutations in the Fusarium solani Species Complex: First Clue to Understand the Low Susceptibility to Azoles of the Genus Fusarium.

Members of Fusarium solani species complex (FSSC) are cosmopolitan filamentous fungi responsible for invasive fungal infections in immunocompromised patients. Despite the treatment recommendations, many strains show reduced sensitivity to voriconazole. The objective of this work was to investigate the potential relationship between azole susceptibility and mutations in CYP51 protein sequences. Minimal inhibitory concentrations (MICs) for azole antifungals have been determined using the CLSI (Clinical and Laboratory Standards Institute) microdilution method on a panel of clinical and environmental strains. CYP51A, CYP51B and CYP51C genes for each strain have been sequenced using the Sanger method. Amino acid substitutions described in multiple azole-resistant Aspergillus fumigatus (mtrAf) strains have been sought and compared with other Fusarium complexes’ strains. Our results show that FSSC exhibit point mutations similar to those described in mtrAf. Protein sequence alignments of CYP51A, CYP51B and CYP51C have highlighted different profiles based on sequence similarity. A link between voriconazole MICs and protein sequences was observed, suggesting that these mutations could be an explanation for the intrinsic azole resistance in the genus Fusarium. Thus, this innovative approach provided clues to understand low azole susceptibility in FSSC and may contribute to improving the treatment of FSSC infection.

Transcription Factor Repurposing Offers Insights into Evolution of Biosynthetic Gene Cluster Regulation.

ABSTRACTThe fungal kingdom has provided advances in our ability to identify biosynthetic gene clusters (BGCs) and to examine how gene composition of BGCs evolves across species and genera. However, little is known about the evolution of specific BGC regulators that mediate how BGCs produce secondary metabolites (SMs). A bioinformatics search for conservation of the Aspergillus fumigatus xanthocillin BGC revealed an evolutionary trail of xan-like BGCs across Eurotiales species. Although the critical regulatory and enzymatic genes were conserved in Penicillium expansum, overexpression (OE) of the conserved xan BGC transcription factor (TF) gene, PexanC, failed to activate the putative xan BGC transcription or xanthocillin production in P. expansum, in contrast to the role of AfXanC in A. fumigatus. Surprisingly, OE::PexanC was instead found to promote citrinin synthesis in P. expansum via trans induction of the cit pathway-specific TF, ctnA, as determined by cit BGC expression and chemical profiling of ctnA deletion and OE::PexanC single and double mutants. OE::AfxanC results in significant increases of xan gene expression and metabolite synthesis in A. fumigatus but had no effect on either xanthocillin or citrinin production in P. expansum. Bioinformatics and promoter mutation analysis led to the identification of an AfXanC binding site, 5′-AGTCAGCA-3′, in promoter regions of the A. fumigatus xan BGC genes. This motif was not in the ctnA promoter, suggesting a different binding site of PeXanC. A compilation of a bioinformatics examination of XanC orthologs and the presence/absence of the 5′-AGTCAGCA-3′ binding motif in xan BGCs in multiple Aspergillus and Penicillium spp. supports an evolutionary divergence of XanC regulatory targets that we speculate reflects an exaptation event in the Eurotiales.

Automated Quantitative Analysis of Airway Epithelial Cell Detachment Upon Fungal Challenge.

Host-pathogen interactions involve a complex interplay between host and pathogen factors, resulting in either host protective immunity or establishment of disease. One of the hallmarks for disease progression is host tissue destruction. The first host surface to interact with the opportunistic respiratory fungal pathogen, Aspergillus fumigatus, is the airway epithelium. Unravelling the mechanisms involved in airway epithelial cell damage by A. fumigatus is essential to understanding the establishment and progression of infection in the host. Although host cell damage can be measured in vitro by indirect cell lysis assays, here, we describe an automated, simple, and low-cost assay to directly visualize and quantify epithelial cell line damage after challenge with A. fumigatus. We employ the previously characterized tissue noninvasive A. fumigatus ΔpacC mutant to demonstrate the quantitative difference in cell damage relative to its parental tissue invasive strain. This assay is easily scaled up for high-throughput screening of multiple Aspergillus mutants and can be adapted to suit diverse host cell lines, different time points of infection, challenge with other microbes, and drugs or novel compounds.

A copper monosulfide-nanoparticle-based fluorescent probe for the sensitive and specific detection of ochratoxin A

The mycotoxin ochratoxin A (OTA) is a secondary metabolite derived from multiple Aspergillus and Penicillium strains. The development of a rapid, sensitive, and simple method for OTA detection is important to ensure food biosafety and safeguard public health. In this study, we designed a highly specific and sensitive assay for the detection of OTA using copper monosulfide (CuS) nanoparticles conjugated to an anti-OTA antibody (CuS-Ab NPs) and a fluorescent probe for Cu2+. When OTA is present in the solution, the OTA antigen, bound to the microplate, is competed off by the soluble OTA for binding to CuS-Ab NPs. After washing, the CuS-Ab NPs and bound OTA are removed. Subsequently, HCl is added to dissolve the CuS-Ab NPs bound to the OTA antigen, releasing Cu2+ and activating the Cu2+ fluorescent probe. Thus, the resultant fluorescence emission is inversely proportional to the OTA content in the solution. Under optimal conditions, this method detected 0.1–100 ng mL−1 OTA with a limit of detection of 0.01 ng mL−1. The assay was tested using corn, soybean, and coffee samples, with recoveries ranging from 94% to 110%. This strategy provides a new approach for the detection of mycotoxins and other small-molecule analytes with broad application potential in food safety and quality control.

Underlying Conditions and Clinical Spectrum of Chronic Pulmonary Aspergillosis (CPA): An Experience from a Tertiary Care Hospital in Karachi, Pakistan.

The incidence of chronic pulmonary aspergillosis (CPA) is especially increasing in high tuberculosis (TB) burden countries. Despite a high estimated CPA burden in Pakistan, actual data on CPA are not available. The aim of the current study is to determine the underlying conditions and clinical spectrum of CPA at a tertiary care hospital in Karachi, Pakistan. This is a retrospective chart review study in patients admitted with CPA from January 2012 to December 2017. A total of 67 patients were identified during the study period. Mean age of CPA patients was 45.9 ± 15 years, 44 (65.7%) were male and 19 (28.4%) had diabetes. The most common type of CPA was simple aspergilloma (49.2%) followed by chronic cavitary pulmonary aspergillosis (CCPA) (44.7%). TB was the underlying cause of CPA in 58 (86.6%) patients followed by bronchiectasis caused by allergic bronchopulmonary aspergillosis (ABPA) 8 (11.9%). Aspergillus flavus was identified in 17 (47.2%), followed by A. fumigatus in 13 (36.1%) CPA patients. Isolation of multiple Aspergillus species was found in 10 (25.6%) patients. Itraconazole was given in 27 (40.3%) patients and a combination therapy of itraconazole and surgery was given in 21 (31.34%) patients. We found aspergilloma and CCPA as the most prevalent forms of CPA in our setting. Further large prospective studies using Aspergillus specific immunoglobulin G (IgG) antibodies testing are required for better understanding of CPA in Pakistan.

Multiple invasive aspergillus brain abscess in an immunocompetent patient without any history of trauma

Aspergillus is a fungus found in the environment. In an immunocompetent person, inhalation of spores may cause localized infection. In immune compromised patients, these fungi can cause life-threatening invasive infections which has high morbidity and mortality. Invasive aspergillosis has a poor prognosis. Intracranial aspergillosis is an extremely rare manifestation of invasive aspergillosis in immunocompetent individuals. A case of 60-year-old immunocompetent male is reported who had multiple Aspergillus brain abscess.

In Vitro Interactions between Target of Rapamycin Kinase Inhibitor and Antifungal Agents against Aspergillus Species.

In vitro interactions of INK128, a target of rapamycin (TOR) kinase inhibitor, and antifungals, including itraconazole, voriconazole, posaconazole, amphotericin B, and caspofungin, against Aspergillus spp. were assessed with the broth microdilution checkerboard technique. Our results suggested synergistic effects between INK128 and all azoles tested, against multiple Aspergillus fumigatus and Aspergillus flavus isolates. However, no synergistic effects were observed when INK128 was combined with amphotericin B or caspofungin. No antagonism was observed for any combination.

Multiple Fungal Brain Abscesses in a Child with Acute Lymphoblastic Leukemia

Fungal infection is a severe problem in children suffering from cancer. We report a case of a four-year-old girl who was diagnosed with acute lymphoblastic leukemia and multiple Aspergillus niger abscesses at the induction phase of the treatment. She was treated with granulocyte transfusions, liposomal amphotericin B with a combination of voriconazole for four months, followed by oral variconazole alone for 17 months. She was successfully treated with this combination without any sequel, and the planned chemotherapy was also completed. Our experience revealed that antifungal treatment including intravenous amphotericin B and variconazole augmented by granulocyte transfusion is an alternative option for the management of this catastrophic complication.

Interrogation of related clinical pan-azole-resistant Aspergillus fumigatus strains: G138C, Y431C, and G434C single nucleotide polymorphisms in cyp51A, upregulation of cyp51A, and integration and activation of transposon Atf1 in the cyp51A promoter.

Multiple Aspergillus fumigatus isolates from a patient with two aspergillomas complicating chronic pulmonary aspergillosis were pan-azole resistant. Microsatellite typing was identical for all isolates despite major phenotypic and some growth rate differences. Three different cyp51A mutations were found (G138C, Y431C, and G434C), of which the first two were demonstrated by heterologous expression in a hypersusceptible Saccharomyces cerevisiae strain to be at least partly responsible for elevated MICs. cyp51A and cyp51B gene duplication was excluded, but increased expression of cyp51A was demonstrated in three isolates selected for additional study (7-to 13-fold increases). In the isolate with the greatest cyp51A expression, an Aft1 transposon was found inserted 370 bp upstream of the start codon of the cyp51A gene, an integration location never previously demonstrated in Aspergillus. Two transcription start sites were identified at 49 and 136 bp upstream of the start codon. The role of the Aft1 transposon, if any, in modulating cyp51A expression remains to be established. Increased mRNA expression of the transporters AfuMDR1 and AfuMDR4 also was demonstrated in some isolates, which could contribute to azole resistance or simply represent a stress response. The diversity of confirmed and possible azole resistance mechanisms demonstrated in a single series of isogenic isolates is remarkable, indicating the ability of A. fumigatus to adapt in the clinical setting.

Multiple Aspergillus Cerebellar Abscesses in a Middle-Aged Female: Case Report and Literature Review

Aspergillus abscesses in the cerebellum are extremely rare, and most cases are solitary. Here, we report the first case of multiple Aspergillus cerebellar abscesses in a 46-year-old female after one mastoidectomy, two craniectomies, and extended use of antibiotics. The possible pathogenesis of this unusual event is discussed. Good outcome was achieved by treatment with a combination of neurosurgical resection and voriconazole (VRC) administration, which we suggest is a potential management plan.

Fungal species identification from avian lung specimens by single hypha laser microdissection and PCR product sequencing

Accurate species diagnosis in cases of fungal pneumonia may be hampered by environmental contamination and colonization resulting in false-positive results. Our novel approach for fungal species diagnostics combines fluorescent staining of mounted cryosections with the optical brightener Blankophor, laser capture microdissection and PCR amplification with subsequent sequencing of the first internal transcribed spacer region (ITS-1). Using clinical specimens from infected birds, we show that the procedure is suitable for species identification from single hyphae of intralesional filamentous fungi. Our data also suggest that multiple Aspergillus fumigatus strain infections may occur frequently in pulmonary aspergillosis of birds.

The Aspergillus Genome Database, a curated comparative genomics resource for gene, protein and sequence information for the Aspergillus research community

The Aspergillus Genome Database (AspGD) is an online genomics resource for researchers studying the genetics and molecular biology of the Aspergilli. AspGD combines high-quality manual curation of the experimental scientific literature examining the genetics and molecular biology of Aspergilli, cutting-edge comparative genomics approaches to iteratively refine and improve structural gene annotations across multiple Aspergillus species, and web-based research tools for accessing and exploring the data. All of these data are freely available at http://www.aspgd.org. We welcome feedback from users and the research community at aspergillus-curator@genome.stanford.edu.

Treatment for Multiple Aspergillus Spondylitis Including a Hip Joint

Multiple aspergillus spondylitis (AS) is a life threatening infection that occurs more commonly in immunocompromised patients, and is commonly treated with antifungal agents. However, there is relatively little information available on the treatment of multiple AS. The authors encountered a 46-year-old man suffering from low back and neck pain with radiculomyelopathy after a liver transplant. The patient had concomitant multiple AS in the cervico-thoraco-lumbar spine and right hip joint, as confirmed by radiologic imaging studies. The pathological examination of a biopsy specimen revealed fungal hyphae at the cervical and lumbar spine. Anterior decompression and interbody fusion were performed for the cervical and lumbar lesions, which showed instability and related neurological symptoms. Additional antifungal therapy was also performed. The patient was treated successfully with remission of his symptoms.

Initial experience in adult-to-adult living donor liver transplantation

Objective To summarize the initial experience in adult-to-adult living donor liver transplantation(ALDLT),so as to improve the efficacy of ALDLT.Methods The clinical data of 31 adult patients who undelwent ALDLT from June 2006 to February 2008 were retrospectively analyzed.Results Of all the patients,8 was with decompensated cirrhosis,7 with acute liver failure,12 with hepatocellular carcinoma,2 with purpura of liver,1 with hilar cholangiocarcinoma,and 1 with Wilson disease.The liver grafts included 25 right lobes with middle hepatic vein(MHV),3 right lobes without MHV,1 right lobe with MHV+left lateral lobe,1 right lobe with MHV+left lobe.The remaining 1 patient underwent auxiliary partial orthotopic liver transplantation with left lobe graft.Six post-transplantation complications occurred in 5 donors. Eleven post-transplantation complications occurred in 9 recipients,including 4 biliary complications,3 vascular complications,3 infection complications and 1 delayed healing of the incision.After ALDLT,2 recipients died of pulmonary infection and 1 of multiple aspergillus infection. Conclusions ALDLT has become an effective method to expand the source of liver grafts.Rational donor and recipient assessment,surgical procedure and postoperative management are key to ALDLT. Key words: Liver transplantation; Living donor

Multiple Aspergillus brain abscesses in immuno-competent patient with severe cranio-facial trauma

Aspergillosis of the central nervous system (CNS) is a rare, but well described disease in immuno-competent patients. We present a 65-year-old patient who developed neuro-aspergillosis 10 months after severe cranio-facial trauma (Le Fort III). He was treated successfully with surgery including stereotactic drainage and, with Amphotericin B, Liposomal Amphotericin B, and Itraconazol.

Whole genome comparison of the A. fumigatus family.

The availability of the genome sequences of multiple Aspergillus spp. presents the research community with an unprecedented opportunity for discovery. The genomes of Neosartorya fischeri and Aspergillus clavatus have been sequenced in order to extend our knowledge of Aspergillus fumigatus, the primary cause of invasive aspergillosis. Through comparative genomic analysis, we hope to elucidate both obvious and subtle differences between genomes, developing new hypotheses that can be tested in the laboratory. A preliminary examination of the genomes and their predicted proteomes reveals extensive conservation between protein sequences and significant synteny, or conserved gene order. Comparative genomic analysis at the level of these closely related aspergilli should provide important insight into the evolutionary forces at play and their effect on gene content, regulation and expression.