Pseudomonas and aspergillus symbiotic coinfections in a case of chronic obstructive pulmonary disease and diabetes mellitus.

Coinfection of Pseudomonas and Aspergillus has not been previously reported in patients with chronic obstructive pulmonary disease (COPD). A middle-aged, thinly built woman (Body Mass Index: 18.1 kg/m²) who smokes bidi (a type of tobacco) and has a history of exposure to open log fires for cooking, has been suffering from COPD for the last 4 years. She has been taking inhaled betamethasone and tiotropium. Additionally, she had uncontrolled diabetes for a few months. She presented with fever, productive cough, shortness of breath and chest pain for 5 days. She required non-invasive ventilation support for type-2 respiratory failure. Chest X-ray and CT confirmed pneumonia, cavities and abscesses in both lungs. Repeated sputum and bronchoalveolar lavage confirmed coinfections with Pseudomonas aeruginosa and Aspergillus fumigatus, respectively. Along with supportive therapy, she was treated with tablet levofloxacin and injection amikacin for 6 weeks based on culture sensitivity reports, and capsule itraconazole for 6 months. She recovered completely to her baseline COPD and diabetes status. This case study confirms that coinfections can occur in COPD and diabetes, highlighting the need for clinicians to be vigilant for the possibility of such symbiotic coinfections.

Aspergillus fumigatus Postoperative Fasciitis and Peritonitis.

A 67 year-old male was admitted in the ICU because of multi-organ failure due to sepsis secondary to Fournier's gangrene. He had sustained radical prostatectomy in the last 48 hours. Peritoneal fluid and fatty tissue biopsies grew Aspergillus Fumigatus without concomitant pulmonary involvement. Postoperative acquisition via exogenous and endogenous routes is discussed, as this nosocomial entity is very rarely reported apart from peritoneal dialysis, especially in non-immunosuppressed patients.

Azole Resistance in Veterinary Clinical Aspergillus fumigatus Isolates in the Netherlands.

Aspergillus fumigatus is a saprophytic fungal pathogen that causes opportunistic infections in animals and humans. Azole resistance has been reported globally in human A. fumigatus isolates, but the prevalence of resistance in isolates from animals is largely unknown. A retrospective resistance surveillance study was performed using a collection of clinical A. fumigatus isolates from various animal species collected between 2015 and 2020. Agar-based azole resistance screening of all isolates was followed by in vitro antifungal susceptibility testing and cyp51A gene sequencing of the azole-resistant isolates. Over the 5 year period 16 (11.3%) of 142 A. fumigatus culture-positive animals harbored an azole-resistant isolate. Resistant isolates were found in birds (15%; 2/13), cats (21%; 6/28), dogs (8%; 6/75) and free-ranging harbor porpoise (33%; 2/6). Azole-resistance was cyp51A mediated in all isolates: 81.3% (T-67G/)TR34/L98H, 12.5% TR46/Y121F/T289A. In one azole-resistant A. fumigatus isolate a combination of C(-70)T/F46Y/C(intron7)T/C(intron66)T/M172V/E427K single-nucleotide polymorphisms in the cyp51A gene was found. Of the animals with an azole-resistant isolate and known azole exposure status 71.4% (10/14) were azole naive. Azole resistance in A. fumigatus isolates from animals in the Netherlands is present and predominantly cyp51A TR-mediated, supporting an environmental route of resistance selection. Our data supports the need to include veterinary isolates in resistance surveillance programs. Veterinarians should consider azole resistance as a reason for therapy failure when treating aspergillosis and consider resistance testing of relevant isolates.

Metagenomic next-generation sequencing could play a pivotal role in validating the diagnosis of invasive mold disease of the central nervous system.

Background: Invasive mold diseases of the central nervous (CNS IMD) system are exceedingly rare disorders, characterized by nonspecific clinical symptoms. This results in significant diagnostic challenges, often leading to delayed diagnosis and the risk of misdiagnosis for patients. Metagenomic Next-Generation Sequencing (mNGS) holds significant importance for the diagnosis of infectious diseases, especially in the rapid and accurate identification of rare and difficult-to-culture pathogens. Therefore, this study aims to explore the clinical characteristics of invasive mold disease of CNS IMD in children and assess the effectiveness of mNGS technology in diagnosing CNS IMD. Methods: Three pediatric patients diagnosed with Invasive mold disease brain abscess and treated in the Pediatric Intensive Care Unit (PICU) of the First Affiliated Hospital of Zhengzhou University from January 2020 to December 2023 were selected for this study. Results: Case 1, a 6-year-old girl, was admitted to the hospital with "acute liver failure." During her hospital stay, she developed fever, irritability, and seizures. CSF mNGS testing resulted in a negative outcome. Multiple brain abscesses were drained, and Aspergillus fumigatus was detected in pus culture and mNGS. The condition gradually improved after treatment with voriconazole combined with caspofungin. Case 2, a 3-year-old girl, was admitted with "acute B-lymphoblastic leukemia." During induction chemotherapy, she developed fever and seizures. Aspergillus fumigatus was detected in the intracranial abscess fluid by mNGS, and the condition gradually improved after treatment with voriconazole combined with caspofungin, followed by "right-sided brain abscess drainage surgery." Case 3, a 7-year-old girl, showed lethargy, fever, and right-sided limb weakness during the pending chemotherapy period for acute B-lymphoblastic leukemia. Rhizomucor miehei and Rhizomucor pusillus was detected in the cerebrospinal fluid by mNGS. The condition gradually improved after treatment with amphotericin B combined with posaconazole. After a six-month follow-up post-discharge, the three patients improved without residual neurological sequelae, and the primary diseases were in complete remission. Conclusion: The clinical manifestations of CNS IMD lack specificity. Early mNGS can assist in identifying the pathogen, providing a basis for definitive diagnosis. Combined surgical treatment when necessary can help improve prognosis.

Detection of pan-azole resistant Aspergillus fumigatus in horticulture and a composting facility in Belgium.

Azole resistance in Aspergillus fumigatus (ARAf) is becoming a worldwide health threat due to increasing occurrence in the environment. However, environmental surveillance programs are not commonly in place and are lacking in Belgium. Since no data on the occurrence of ARAf and the presence of hotspots for the selection of azole resistance is available in Belgium, a first study on the prevalence of ARAf in the environment was conducted. A total of 232 air and compost or soil samples were taken from two composting facilities, and from horticultural and agricultural crops. The azole susceptibility pattern was determined using the EUCAST method (E. Def. 9.4), and the cyp51A gene and its promotor region were sequenced in A. fumigatus isolates with phenotypic azole resistance. Six pan-azole-resistant A. fumigatus isolates were identified, originating from compost and horticultural crops. Four isolates carried the TR34/L98H mutation, and one isolate carried the TR46/Y121F/T289A mutation. However, we did not observe any ARAf isolates from agricultural crops. In conclusion, this study reported the first TR34/L98H and TR46/Y121F/T289A mutation isolated from a composting facility and horticulture in Belgium. The implementation of standardization in environmental surveillance of A. fumigatus on a European level would be beneficial in order to identify hotspots.

The ubiquitous fungus Aspergillus fumigatus can cause serious invasive diseases in humans. Due to the extensive use of environmental azoles, an increase of clinical infections with azole-resistant A. fumigatus is seen. This pilot study aimed to estimate the prevalence of azole-resistant A. fumigatus in environmental reservoirs in Belgium.

First detection of triazole-resistant aspergillus fumigatus harbouring the TR34/L98H Cyp51A mutation in Burkina Faso.

BACKGROUND: Triazole-resistant Aspergillus fumigatus (TRAF) isolates are a growing public health problem with worldwide distribution. Epidemiological data on TRAF is limited in Africa, particularly in West Africa. OBJECTIVES: This study aimed to screen for the environmental presence of TRAF isolates in the indoor air of two hospitals in Burkina Faso. MATERIALS AND METHODS: Air samples were collected in wards housing patients at risk for invasive aspergillosis, namely infectious diseases ward, internal medicine ward, nephrology ward, pulmonology ward, medical emergency ward and paediatric ward. Sabouraud Dextrose Agar supplemented with triazoles was used to screen the suspected TRAF isolates and EUCAST method to confirm the resistance of suspected isolates. Sequencing of cyp51A gene was used to identify the resistance mechanism of confirmed TRAF isolates. RESULTS: Of the 198 samples collected and analysed, 67 showed growth of A. fumigatus isolates. The prevalence of TRAF isolates was 3.23% (4/124). One TRAF isolate exhibited a pan-triazole resistance. Sequencing of cyp51A gene identified the TR34/L98H mutation for this pan-triazole resistant isolate. This study showed for the first time the circulation of the pan-azole resistant isolate harbouring the TR34/L98H mutation in Burkina Faso. CONCLUSIONS: These findings emphasise the need to map these TRAF isolates in all parts of Burkina Faso and to establish local and national continuous surveillance of environmental and clinical TRAF isolates in this country.

Comparison of different lateral flow assays on bronchoalveolar lavage fluid for invasive aspergillosis screening in non-hematological patients.

Several lateral flow assays (LFA) capable of detecting Aspergillus fumigatus in serum and broncho-alveolar lavage fluid (BALF) within the hour, thereby potentially accelerating the screening process, are now commercially available. We prospectively compared three LFA targeting A. fumigatus on BALF collected from non-surgical intensive care patients between June 2022 and February 2023. The three LFA tested were Sõna Aspergillus galactomannan LFA (Immy), Fungadia Aspergillus antigen (Gadia), and AspLFD (OLM Diagnostics). We compared the results of these LFA with those of the galactomannan (GM) Platelia Aspergillus enzyme immunoassay (Bio-Rad), culture on Sabouraud medium and Aspergillus qPCR. We tested 97 BALF samples from 92 patients. In total 84 BALF samples tested negative with all three LFA, and four BALF samples tested positive with the AspLFD assay only (OLM). Only one BALF sample tested positive with the three LFA. In addition, three BALF samples tested positive only with the GM Platelia immunoassay. Four diagnosis of probable invasive aspergillosis were retained for the 92 patients tested. This prospective series included very few positive samples. From a practical point of view, the LFA from OLM presented the simplest protocol for use.

Pathogenic potential of an environmental Aspergillus fumigatus strain recovered from soil of Pygoscelis papua (Gentoo penguins) colony in Antarctica.

Aspergillus fumigatus is a common opportunistic pathogen in different animals, including birds such as penguins. For the first time, a fungal strain identified as A. fumigatus was isolated from soil in the nests of gentoo penguins, Pygoscelis papua, on Livingston Island, South Shetland Islands (maritime Antarctica). This isolate (A. fumigatus UFMGCB 11829) displayed a series of potentially pathogenic characteristics in vitro. We evaluated its detailed molecular taxonomy and submitted the A. fumigatus UFMGCB 11829 Antarctic strain to in vivo pathogenic modelling. The isolate was confirmed to represent A. fumigatus morphological and phylogenetic analysis showed that it was closely related to A. fumigatus sequences reported from animals, immunosuppressed humans, storage grains, plants and soils. The strain displayed the best mycelial growth and conidia production at 37 ºC; however, it was also able to grow and produce conidia at 15º, demonstrating its capability to survive and colonize penguin nest at least in the summer season in maritime Antarctica. In pathogenicity tests, healthy mice did not showed symptoms of infection; however, 50% lethality was observed in immunosuppressed mice that were inoculated with 106 and 107 spores. Lethality increased to 100% when inoculated with 108 spores. Our data highlight the potential pathogenicity of opportunistic A. fumigatus that may be present in the Antarctic, and the risks of both their further transfer within Antarctica and outwards to other continents, risks which may be exacerbated due global climatic changes.

Is there an interest in systematic serum screening for aspergillosis in COVID-19 patients in a medical ward?

PURPOSE: We evaluated the interest of systematic screening of serum fungal markers in patients hospitalized in a medical ward. METHODS: We retrospectively analyzed all patients hospitalized in our infectious disease department from October 1st to October 31st, 2020 for COVID-19 without prior ICU admission, and for whom systematic screening of serum fungal markers was performed. RESULTS: Thirty patients were included. The majority of patients received corticosteroids (96.7%). The galactomannan antigen assay was positive for 1/30 patients at D0, and 0/24, 0/16, 0/13 and 0/2 at D4, D7, D10 and D14 respectively. 1,3-ß-D-glucan was positive for 0/30, 1/24, 1/12, 0/12, 0/2 at D0, D4, D7, D10 and D14 respectively. No Aspergillus fumigatus PCR was positive. No cases of aspergillosis were retained. CONCLUSION: Our study does not support the interest of systematic screening of fungal markers in immunocompetent patients with COVID-19 in a conventional unit.

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

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

Longitudinally extensive posterolateral myelitis revealing invasive CNS aspergillosis in an immunocompetent patient.

BACKGROUND: Fungal infections of the central nervous system usually affect immunocompromised patients. Primary Aspergillus myelitis has never been described. REPORT: A 45-year-old immunocompetent male with subacute paraplegia was treated for inflammatory myelitis before clinical deterioration requiring mechanical ventilation. Purulent meningitis preceded the formation of a paraspinal nodule biopsied by neurosurgery. Histopathological analysis revealed the presence of fungal hyphae, and polymerase chain reaction was positive for Aspergillus fumigatus. No cause of immunodeficiency was identified in this patient. DISCUSSION: Primary Aspergillus myelitis may be confused with inflammatory myelitis and should be considered even in the absence of apparent immunosuppression.

Chromatin profiling reveals heterogeneity in clinical isolates of the human pathogen Aspergillus fumigatus.

Invasive Pulmonary Aspergillosis, which is caused by the filamentous fungus Aspergillus fumigatus, is a life-threatening infection for immunosuppressed patients. Chromatin structure regulation is important for genome stability maintenance and has the potential to drive genome rearrangements and affect virulence and pathogenesis of pathogens. Here, we performed the first A. fumigatus global chromatin profiling of two histone modifications, H3K4me3 and H3K9me3, focusing on the two most investigated A. fumigatus clinical isolates, Af293 and CEA17. In eukaryotes, H3K4me3 is associated with active transcription, while H3K9me3 often marks silent genes, DNA repeats, and transposons. We found that H3K4me3 deposition is similar between the two isolates, while H3K9me3 is more variable and does not always represent transcriptional silencing. Our work uncovered striking differences in the number, locations, and expression of transposable elements between Af293 and CEA17, and the differences are correlated with H3K9me3 modifications and higher genomic variations among strains of Af293 background. Moreover, we further showed that the Af293 strains from different laboratories actually differ in their genome contents and found a frequently lost region in chromosome VIII. For one such Af293 variant, we identified the chromosomal changes and demonstrated their impacts on its secondary metabolites production, growth and virulence. Overall, our findings not only emphasize the influence of genome heterogeneity on A. fumigatus fitness, but also caution about unnoticed chromosomal variations among common laboratory strains.

Phylogenetic Distribution of csp1 Types in Aspergillus fumigatus and Their Correlates to Azole Antifungal Drug Resistance.

In Aspergillus fumigatus, the repetitive region of the csp1 gene is one of the most frequently used loci for intraspecies typing of this human pathogenic mold. Using PCR amplification and Sanger sequencing of only a single marker, csp1 typing is readily available to most laboratories and highly reproducible. Here, I evaluate the usefulness of the csp1 marker for resistance detection and epidemiologic stratification among A. fumigatus isolates. After resolving nomenclature conflicts from published studies and adding novel csp1 types, the number of known types now adds up to 38. Their distribution mostly correlates with A. fumigatus population structure, and they are also meaningful for narrowly defined cases of azole resistance phenotypes. Isolates carrying the pandemic resistance allele TR34/L98H show signs of interclade crossing of strains with t02 or t04A, into the t11 clade. Furthermore, absolute differences in voriconazole MIC values between t02/t04B versus t11 TR34/L98H isolates indicate that the genetic background of resistance mutations may have a pivotal role in cross-resistance phenotypes and, thus, clinical outcome and environmental selection. Despite the general genetic similarity of isolates with identical csp1 types, outcrossing into other clades is also observed. The csp1 type alone, therefore, does not sufficiently discriminate genetic clades to be used as the sole marker in epidemiologic studies. IMPORTANCE Aspergillus fumigatus is a ubiquitously distributed saprophytic mold and a leading cause of invasive aspergillosis in human hosts. Pandemic azole-resistant strains have emerged on a global scale, which are thought to be propagated through use of azole-based fungicides in agriculture. To perform epidemiologic studies, genetic typing of large cohorts is key. Here, I evaluate the usefulness of the frequently used csp1 marker for resistance detection and epidemiologic stratification among A. fumigatus isolates. The phylogenetic distribution of csp1 types mostly correlates with A. fumigatus population structure and is also meaningful for narrowly defined cases of azole resistance phenotypes. Nevertheless, outcrossing of csp1 into other clades is also observed. The csp1 type alone, therefore, does not sufficiently discriminate genetic clades and should not be used as the sole marker in epidemiologic studies.

Aspergillus fumigatus pan-genome analysis identifies genetic variants associated with human infection.

Aspergillus fumigatus is an environmental saprobe and opportunistic human fungal pathogen. Despite an estimated annual occurrence of more than 300,000 cases of invasive disease worldwide, a comprehensive survey of the genomic diversity present in A. fumigatus-including the relationship between clinical and environmental isolates and how this genetic diversity contributes to virulence and antifungal drug resistance-has been lacking. In this study we define the pan-genome of A. fumigatus using a collection of 300 globally sampled genomes (83 clinical and 217 environmental isolates). We found that 7,563 of the 10,907 unique orthogroups (69%) are core and present in all isolates and the remaining 3,344 show presence/absence of variation, representing 16-22% of the genome of each isolate. Using this large genomic dataset of environmental and clinical samples, we found an enrichment for clinical isolates in a genetic cluster whose genomes also contain more accessory genes, including genes coding for transmembrane transporters and proteins with iron-binding activity, and genes involved in both carbohydrate and amino-acid metabolism. Finally, we leverage the power of genome-wide association studies to identify genomic variation associated with clinical isolates and triazole resistance as well as characterize genetic variation in known virulence factors. This characterization of the genomic diversity of A. fumigatus allows us to move away from a single reference genome that does not necessarily represent the species as a whole and better understand its pathogenic versatility, ultimately leading to better management of these infections.

Granulomatous fungal and non-tuberculous mycobacterial infestation complicating chronic lung disease: Outcomes in patients undergoing lung transplantation.

INTRODUCTION: Granulomatous infections are common in patients with chronic lung disease. We aim to study the incidence and clinicopathological features of granulomatous infections in a cohort of patients undergoing lung transplantation for end-stage chronic lung disease. METHODS: Pathology reports of 50 explanted native lungs of patients who underwent lung transplantation since 2015 at our institution were reviewed. Four cases with granulomatous lesions were identified. Correlation was made with clinical findings in the 4 cases. RESULTS: The granulomatous infections include non-necrotizing cryptococcal pneumonitis (case 1), necrotizing pneumonia due to Scedosporium sp. and Mycobacterium avium Complex (MAC) (Cases 2 and 3), and invasive Aspergillus pneumonia (Case 4). One patient received pre-transplant fungal prophylaxis (Case 4). Post-transplant infectious complications included invasive (Cases 2 and 4) and non-invasive (Case 1) fungal infections and bacterial pneumonia (Cases 1 and 2). Two patients (Cases 3 and 4) developed acute cellular rejection (ACR) in the first 30 days. The third patient (Case 1) was identified with ACR in the 9 months post-transplant and chronic lung allograft dysfunction at 29 months. In terms of mortality, 1 patient (Case 1) died at 30 months post-transplant from pseudomonal sepsis and chronic graft failure. Two patients with invasive fungal infections (Cases 2 and 4) are on secondary prophylaxis and doing well. One patient (Case 3) remains infection-free and on MAC prophylaxis. CONCLUSIONS: In our case series, patients with chronic lung diseases with superimposed granulomatous infestations frequently experienced post-transplant complications. These include invasive infections and repeat ACRs that predispose patients to chronic graft dysfunction. Pre- and post-transplant antifungal prophylaxis reduces fungal load and complication risk post-transplant.

Are Point Mutations in HMG-CoA Reductases (Hmg1 and Hmg2) a Step towards Azole Resistance in Aspergillus fumigatus?

Invasive aspergillosis, mainly caused by Aspergillus fumigatus, can lead to severe clinical outcomes in immunocompromised individuals. Antifungal treatment, based on the use of azoles, is crucial to increase survival rates. However, the recent emergence of azole-resistant A. fumigatus isolates is affecting the efficacy of the clinical therapy and lowering the success rate of azole strategies against aspergillosis. Azole resistance mechanisms described to date are mainly associated with mutations in the azole target gene cyp51A that entail structural changes in Cyp51A or overexpression of the gene. However, strains lacking cyp51A modifications but resistant to clinical azoles have recently been detected. Some genes have been proposed as new players in azole resistance. In this study, the gene hmg1, recently related to azole resistance, and its paralogue hmg2 were studied in a collection of fifteen azole-resistant strains without cyp51A modifications. Both genes encode HMG-CoA reductases and are involved in the ergosterol biosynthesis. Several mutations located in the sterol sensing domain (SSD) of Hmg1 (D242Y, G307D/S, P309L, K319Q, Y368H, F390L and I412T) and Hmg2 (I235S, V303A, I312S, I360F and V397C) were detected. The role of these mutations in conferring azole resistance is discussed in this work.