Rasamsonia Argillacea Research Articles

Detection of Specific IgE against Molds Involved in Allergic Bronchopulmonary Mycoses in Patients with Cystic Fibrosis.

Allergic bronchopulmonary mycoses (ABPM) can be due to molds other than Aspergillus fumigatus in patients with cystic fibrosis (pwCF). We aimed to develop immunoassays for the detection of specific IgE (sIgE) directed against five fungal species involved in ABPM: Aspergillus terreus, Scedosporium apiospermum, Lomentospora prolificans, Rasamsonia argillacea, and Exophiala dermatitidis. Serum samples (n = 356) from 238 pwCF, collected in eight CF care centers in France, Germany, and Italy, were analyzed by dissociated enhanced lanthanide fluorescent immunoassay (DELFIA®) to assess levels of sIgE directed against antigenic extracts of each fungus. Clinical, biological, and radiological data were collected for each episode. One hundred serum samples from healthy blood donors were used as controls. Sera were classified into four groups depending on the level of sIgE according to the quartile repartition calculated for the pwCF population. A score of 4 for values above the 3rd quartile corresponds to an elevated level of sIgE. PwCF showed higher levels of sIgE than controls. Based on criteria from the ABPA-ISHAM working group, with an additional criterion of "a sIgE score of 4 for at least one non-A. fumigatus mold", we were able to diagnose six cases of ABPM. Using 417IU/mL as the threshold for total IgE and the same additional criterion, we identified seven additional pwCF with "putative ABPM". Detection of sIgE by DELFIA® showed good analytical performance and supports the role played by non-A. fumigatus molds in ABPM. However, commercially available kits usable in routine practice are needed to improve the diagnosis of ABPM.

Disseminated Rasamsonia argillacea infection in a dog

ABSTRACT Case history A 4-year-old, male neutered Borzoi presented for unlocalised pain and frequent episodes of vocalisation. Clinical findings Pain was localised to the lumbar spine and radiographs revealed a L3–L4 lesion consistent with discospondylitis. The dog was treated for presumptive bacterial discospondylitis with surgical debridement, spinal stabilisation, and cephalexin. Samples collected from the affected intervertebral disc at the time of surgery revealed lymphoplasmacytic inflammation with no causative agent identified on histopathology or bacterial culture. After an initial period of improvement, signs recurred despite an 8-week antibiotic course, with the development of inappetence, weight loss, polydipsia, and polyuria. Repeat radiographs revealed a new cervical intervertebral lesion, and concurrent pyelonephritis was diagnosed based on blood and urine results. Fungal culture of urine resulted in growth of Rasamsonia argillacea species complex and disseminated fungal disease was clinically diagnosed. Antifungal treatment was commenced, however the dog deteriorated, and euthanasia was performed. Pathological findings Multifocal white plaques were grossly visualised in the spleen, mesenteric lymph nodes, cervical vertebrae, and kidneys. Periodic acid-Schiff-positive, fine, parallel-walled, occasionally branching, septate hyphae 5–10 μm in diameter, and conidia 5–7 μm in diameter were found on sectioning all organs. R. argillacea species complex was identified by fungal culture of urine and was considered the species of fungal organism seen histologically. The isolate was subsequently confirmed as R. argillacea by DNA sequencing. Diagnosis Disseminated Rasamsonia argillacea infection. Clinical relevance Rasamsonia argillacea species complex is a recognised invasive mycosis in veterinary medicine, with disseminated disease causing significant clinical complications and death. This is believed to be the first report of infection caused by R. argillacea in a dog in Australasia and highlights the importance of awareness of a potential fungal aetiology in dogs with discospondylitis. Abbreviations: CLSI: Clinical and Laboratory Standards Institute; CRI: Constant rate infusion; MEC: Minimum effective concentration; MIC: Minimum inhibitory concentration; PAS: Periodic acid-Schiff

Non-Aspergillus Hyaline Molds: A Host-Based Perspective of Emerging Pathogenic Fungi Causing Sinopulmonary Diseases.

The incidence of invasive sino-pulmonary diseases due to non-Aspergillus hyaline molds is increasing due to an enlarging and evolving population of immunosuppressed hosts as well as improvements in the capabilities of molecular-based diagnostics. Herein, we review the following opportunistic pathogens known to cause sinopulmonary disease, the most common manifestation of hyalohyphomycosis: Fusarium spp., Scedosporium spp., Lomentospora prolificans, Scopulariopsis spp., Trichoderma spp., Acremonium spp., Paecilomyces variotii, Purpureocillium lilacinum, Rasamsonia argillacea species complex, Arthrographis kalrae, and Penicillium species. To facilitate an understanding of the epidemiology and clinical features of sino-pulmonary hyalohyphomycoses in the context of host immune impairment, we utilized a host-based approach encompassing the following underlying conditions: neutropenia, hematologic malignancy, hematopoietic and solid organ transplantation, chronic granulomatous disease, acquired immunodeficiency syndrome, cystic fibrosis, and healthy individuals who sustain burns, trauma, or iatrogenic exposures. We further summarize the pre-clinical and clinical data informing antifungal management for each pathogen and consider the role of adjunctive surgery and/or immunomodulatory treatments to optimize patient outcome.

Dihydroorotate dehydrogenase inhibitor olorofim has potent in vitro activity against Microascus/Scopulariopsis, Rasamsonia, Penicillium and Talaromyces species.

Treatment options against infections caused by rare but emerging moulds may be limited by their reduced susceptibility or resistance to clinically available antifungals. The investigational antifungal olorofim, which targets the biosynthesis of pyrimidines within fungi, has activity against different species of filamentous fungi, including Aspergillus and Scedosporium/Lomentospora prolificans isolates that are resistant to available antifungals. We evaluated the in vitro activity of olorofim against 160 isolates within the genera Microascus/Scopulariopsis, Penicillium, Talaromyces and the Rasamsonia argillacea species complex. One hundred sixty clinical isolates that had previously been identified to the species level by DNA sequence analysis were included. Antifungal susceptibility testing was performed by CLSI M38 broth microdilution for olorofim, amphotericin B, caspofungin, posaconazole and voriconazole. Olorofim demonstrated in vitro activity against each of the genera tested. Overall, olorofim MICs ranged from ≤0.008 to 0.5mg/L against all isolates tested, with MIC90 and modal MIC values ranging from ≤0.008 to 0.25 mg/L and ≤0.008 to 0.03 mg/L, respectively. This activity was also maintained against individual isolates that had reduced susceptibility to or in vitro resistance against amphotericin B, posaconazole and/or voriconazole. The investigational agent olorofim demonstrated good in vitro activity against clinical isolates of emerging mould pathogens, including those with reduced susceptibility or resistance to clinically available antifungals. Further studies are warranted to determine how well this in vitro activity translates into in vivo efficacy against infections caused by these fungi.

S10.5a Fungal respiratory infections in cystic fibrosis patients in the Middle East

S10.5 Fungal respiratory infections in Cystic Fibrosis, September 24, 2022, 10:30 AM - 12:00 PMCystic Fibrosis (CF) is among the most common genetic disorders, which involves multiple organs including the respiratory tract. CF is thought to be an uncommon disease in the Middle East (ME). However, the prevalence is estimated at 1 in 30 000-50 000, while the incidence is estimated at 1 in 2000-5800 live births. Several studies from ME revealed that many children with CF in these populations probably remain undiagnosed due to lack of clinical suspicion and proper diagnostic facilities. According to the experts’ idea, CF may be more common in Iran than expected before. Chronic colonization of the airways of CF patients and infections due to a wide variety of opportunistic fungal pathogens including Aspergillus, Candida, Scedosporium species, Exophiala dermatitidis, Rasamsonia argillacea complex, and Lomentospora prolificans are currently increasing. On the other hand, the resistance of these opportunistic pathogens to commonly available antifungals challenges therapeutic options and consequently endanger the CF patients’ life. Dissimilar to bacterial colonization or infections, the epidemiology and pathogenicity of colonization and fungal respiratory infections in CF are less known. According to our recent study, the prevalence rate of respiratory colonization was reported as 73.3%. Among mold isolates, Aspergillus was also the most common genus followed by Scedosporium species. In contrast to the reports from western countries, A. flavus was also identified as the most prevalent species of Aspergillus from ME countries including Iran and India. In some studies, allergic bronchopulmonary aspergillosis (ABPA) in CF patients from ME was evaluated. In our recent experience from Iran, of 86 patients with CF, 9 (10.5%) cases were met ABPA diagnosis. A. flavus was the most common agent followed by A. fumigatus, A. terreus and A. tubingensis. A significant resistance of Scedosporium and Aspergillus isolates from CF patients against the main antifungal agents in invasive fungal infections therapy was reported in different studies. According to these realities, there are a few reports on the Research Topic ‘Fungal Respiratory Infections and Colonization in CF’ from Iran and some other ME countries. Therefore, in this presentation, we are going to highlight our experiences and other published data from Iran and ME in this field including clinical presentations, fungal species involved, diagnosis strategies, and in vitro antifungal susceptibility patterns of fungal isolates from CF patients, and common treatments and prophylactic strategies.

Emerging Fungal Infections: New Species, New Names, and Antifungal Resistance.

BackgroundInfections caused by fungi can be important causes of morbidity and mortality in certain patient populations, including those who are highly immunocompromised or critically ill. Invasive mycoses can be caused by well-known species, as well as emerging pathogens, including those that are resistant to clinically available antifungals.ContentThis review highlights emerging fungal infections, including newly described species, such as Candida auris, and those that having undergone taxonomic classification and were previously known by other names, including Blastomyces and Emergomyces species, members of the Rasamsonia argillacea species complex, Sporothrix brasiliensis, and Trichophyton indotinae. Antifungal resistance also is highlighted in several of these emerging species, as well as in the well-known opportunistic pathogen Aspergillus fumigatus. Finally, the increased recognition and importance of fungal co-infections with respiratory pathogens, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is discussed.SummaryBoth clinicians and clinical microbiology laboratories should remain vigilant regarding emerging fungal infections. These may be difficult both to diagnose and treat due to the lack of experience of clinicians and laboratory personnel with these organisms and the infections they may cause. Many of these fungal infections have been associated with poor clinical outcomes, either due to inappropriate therapy or the development of antifungal resistance.

Disseminated Rasamsonia argillacea species complex infections in 8 dogs.

BackgroundClinical features, treatment, and outcome of opportunistic infections with Rasamsonia spp., a nonpigmented filamentous mold, are not well documented in dogs.ObjectivesDescribe clinical, radiographic, pathologic features, and outcome of dogs with disseminated Rasamsonia species complex infections.AnimalsEight client‐owned dogs.MethodsRetrospective case series. Medical records were reviewed to describe signalment, history, clinicopathologic and imaging findings, microbiologic and immunologic results, cyto‐ and histopathologic diagnoses, treatment, and outcome.ResultsPresenting complaints were nonspecific with anorexia (n = 5) and back pain (n = 4) most common. Five dogs were German Shepherd dogs. Six dogs had multifocal discospondylitis and 2 had pleural effusion. Six dogs had Rasamsonia piperina and 2 had Rasamsonia argillacea infections with isolates identified using DNA sequencing. Rasamsonia spp. were isolated by urine culture in 5 of 7 dogs. Five of 6 dogs had positive serum Aspergillus galactomannan antigen enzyme immunoassay (EIA) results. Median survival time was 82 days, and 317 days for dogs that survived to discharge. Four died during initial hospitalization (median survival, 6 days). All isolates had low minimum effective concentrations (MECs) to echinocandins with variable minimum inhibitory concentrations (MICs) for azole antifungal drugs.Conclusions and Clinical ImportanceRasamsonia spp. infections in dogs are associated with multisystemic disease involving the vertebral column, central nervous system, kidneys, spleen, lymph nodes, lungs, and heart. The infection shares clinical features with other systemic mold infections and can be misidentified when using phenotypical microbiologic methods. Molecular techniques are required to identify the organism and guide appropriate antifungal treatment.

ALEMTUZUMAB FOR CHRONIC LUNG ALLOGRAFT DYSFUNCTION

SESSION TITLE: Transplantation Posters SESSION TYPE: Original Investigation Posters PRESENTED ON: October 18-21, 2020 PURPOSE: Chronic lung allograft dysfunction (CLAD) remains the leading cause of long-term mortality after lung transplantation (LTX) with no proven therapeutic strategies except re-transplantation. Anecdotal reports suggest a role for the lymphocyte depleting agent, Alemtuzumab (AL), an anti-CD52 monoclonal antibody. We reviewed our experience with AL for the treatment of refractory CLAD. METHODS: Eight consecutive LTX recipients were identified. AL was given as a single subcutaneous dose of 30 mg. Cell-cycle inhibitor therapy was held and valganciclovir and azole prophylaxis were given for at least 6 months after AL treatment. The slope of FEV1 3 months before and after AL were compared. Complications of AL therapy including infections and survival were assessed. RESULTS: The cohort consisted of 5 males and 3 females with a median age at time of AL administration of 66 y (range: 50-72). Time post-transplant was 2.43 y (range: 1.4-5.4). Pre-transplant diagnoses were COPD (n=3), and 1 each of CF, bronchiectasis (immotile cilia syndrome), IPF, PVOD and IPAH. All subjects were bilateral recipients and 1 was post-left pneumonectomy early after transplant. All had a predominantly obstructive CLAD phenotype, stages 4 (N=2), 3 (N=4), 2 (N=1) and 1 (N=1) with rapid loss of lung function. The median slope of decline in FEV1 in the 3 months prior to AL was -336ml/m (range: -39 to -552) compared with +24 ml/m (range: -171 to +48) during the 3 months post AL administration (P = 0.016). No acute reactions to AL treatment were observed. Clinically symptomatic infections occurred in 4 patients following AL. Community acquired respiratory viral infections were observed in 2 (parainfluenza and coronavirus on 2 separate occasions in 1 patient and rhinovirus in another). Pseudomonas tracheobronchitis developed in 1. These infections were considered mild-moderate. One subject developed new parenchymal opacities with isolation of Rasamsonia argillacea and Mycobacterium fortuitum. Two patients died due to progressive CLAD 3 and 6 months after AL. The other six are alive at a median follow-up time of 12 months (range: 7 – 20). Kaplan-Meier survival estimate at one year was 75%. At last follow-up, CLAD stages among survivors was 4 (N=1), 3 (N=4) and 2 (N=1). CONCLUSIONS: AL therapy was associated with a significant attenuation in lung function decline in lung transplant recipients with rapidly progressive CLAD. Treatment was generally well tolerated with few serious infection complications. CLINICAL IMPLICATIONS: AL should be considered for rapidly progressive CLAD. Randomized controlled trials are required to establish efficacy and safety. DISCLOSURES: No relevant relationships by Reda Girgis, source=Web Response No relevant relationships by Ryan Hadley, source=Web Response no disclosure submitted for Anupam Kumar; No relevant relationships by Cameron Lawson, source=Web Response no disclosure on file for Marzia Leacche; No relevant relationships by Jennifer McDermott, source=Web Response no disclosure on file for Edward Murphy; No relevant relationships by Gayathri Sathiyamoorthy, source=Web Response

In vitro activity of olorofim (F901318) against fungi of the genus, Scedosporium and Rasamsonia as well as against Lomentospora prolificans, Exophiala dermatitidis and azole-resistant Aspergillus fumigatus

In recent decades, invasive infections caused by fungal pathogens have been reported with increasing frequency. Concurrently, the rates of detected resistance mechanisms against commonly used antifungal agents in fungi are increasing. The need for novel antifungal drugs is thus imminent. In this study, the novel drug olorofim (F901318) was tested for its antifungal activity against the human fungal pathogens Lomentospora prolificans (n=20), Scedosporium aurantiacum (n=2), Scedosporium apiospermum (n=6), Rasamsonia argillacea species complex (n=23), Exophiala dermatitidis (n=10) and azole-resistant Aspergillus fumigatus (ARAF) (n=25) in an in vitro broth microdilution assay according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommendations. Whilst olorofim was ascertained to be effective against R. argillacea species complex [minimum inhibitory concentrations (MICs) of ≤0.008 mg/L], Scedosporium spp. (MICs of 0.032-0.5 mg/L), L. prolificans (MICs of 0.032-0.5 mg/L) and ARAF (MICs of ≤0.008-0.032 mg/L), the drug had an MIC of >4 mg/L against E. dermatitidis. These data demonstrate the antifungal activity of olorofim against a broad range of filamentous fungal pathogens.

Non-Aspergillus Fungal Infections in Chronic Granulomatous Disease

Management of Aspergillus infection in chronic granulomatous disease (CGD) patients remains a challenge even in new azoles era. However, epidemiology, diagnosis, and management of non-Aspergillus fungal infection (NAFI) in CGD setting are poorly described. NAFI appears to be rare in CGD patients and receiving antifungal prophylaxis. Clinical presentation is not specific of fungal species and patients often suffer from mild disease with prolonged course. While candidiasis and mucormycosis are also common in other immunocompromised population, infections caused by Phellinus tropicalis, Trichosporon inkin, and Rasamsonia argillacea have a unique predilection for CGD patients. Improved fungal identification with molecular methods allows description of new fungal species. The available data for NAFI during CGD are limited and based on case reports and small series. Invasive tissue biopsies and advanced molecular methods are often required for diagnosis and guiding antifungal strategy in addition to a close collaboration between clinician, pathologist, and mycologist.

Airway persistence by the emerging multi‐azole‐resistant Rasamsonia argillacea complex in cystic fibrosis

Infections caused by Rasamsonia argillacea complex have been reported in various clinical settings. Cystic fibrosis (CF) is one of the main underlying conditions. An observational cohort study of CF patients with Rasamsonia in respiratory samples was conducted. Eight isolates from 6 patients were identified as R.argillacea complex and tested for antifungal susceptibility. All isolates had high MICs to voriconazole and posaconazole and low MECs to echinocandins. Four patients experienced lung function decline in the year preceding first Rasamsonia isolation. This continued in the year following first isolation in 3 out of 4 cases. Antifungal therapy was initiated in 2 patients, to which only one exhibited a clinical response. Three out of 6 patients died within 3years of isolating Rasamsonia. Genotyping suggests that similar genotypes of Rasamsonia can persist in CF airways. Consistent with other fungi in CF, the clinical impact of airway colonisation by Rasamsonia is variable. In certain patients, Rasamsonia may be able to drive clinical decline. In others, though a clear impact on lung function may be difficult to determine, the appearance of Rasamsonia acts as a marker of disease severity. In others it does not appear to have an obvious clinical impact on disease progression.

Developing collaborative works for faster progress on fungal respiratory infections in cystic fibrosis.

Cystic fibrosis (CF) is the major genetic inherited disease in Caucasian populations. The respiratory tract of CF patients displays a sticky viscous mucus, which allows for the entrapment of airborne bacteria and fungal spores and provides a suitable environment for growth of microorganisms, including numerous yeast and filamentous fungal species. As a consequence, respiratory infections are the major cause of morbidity and mortality in this clinical context. Although bacteria remain the most common agents of these infections, fungal respiratory infections have emerged as an important cause of disease. Therefore, the International Society for Human and Animal Mycology (ISHAM) has launched a working group on Fungal respiratory infections in Cystic Fibrosis (Fri-CF) in October 2006, which was subsequently approved by the European Confederation of Medical Mycology (ECMM). Meetings of this working group, comprising both clinicians and mycologists involved in the follow-up of CF patients, as well as basic scientists interested in the fungal species involved, provided the opportunity to initiate collaborative works aimed to improve our knowledge on these infections to assist clinicians in patient management. The current review highlights the outcomes of some of these collaborative works in clinical surveillance, pathogenesis and treatment, giving special emphasis to standardization of culture procedures, improvement of species identification methods including the development of nonculture-based diagnostic methods, microbiome studies and identification of new biological markers, and the description of genotyping studies aiming to differentiate transient carriage and chronic colonization of the airways. The review also reports on the breakthrough in sequencing the genomes of the main Scedosporium species as basis for a better understanding of the pathogenic mechanisms of these fungi, and discusses treatment options of infections caused by multidrug resistant microorganisms, such as Scedosporium and Lomentospora species and members of the Rasamsonia argillacea species complex.

A Phenotypic and Genotypic Analysis of the Antimicrobial Potential of Cultivable Streptomyces Isolated from Cave Moonmilk Deposits.

Moonmilk speleothems of limestone caves host a rich microbiome, among which Actinobacteria represent one of the most abundant phyla. Ancient medical texts reported that moonmilk had therapeutical properties, thereby suggesting that its filamentous endemic actinobacterial population might be a source of natural products useful in human treatment. In this work, a screening approach was undertaken in order to isolate cultivable Actinobacteria from moonmilk of the Grotte des Collemboles in Belgium, to evaluate their taxonomic profile, and to assess their potential in biosynthesis of antimicrobials. Phylogenetic analysis revealed that all 78 isolates were exclusively affiliated to the genus Streptomyces and clustered into 31 distinct phylotypes displaying various pigmentation patterns and morphological features. Phylotype representatives were tested for antibacterial and antifungal activities and their genomes were mined for secondary metabolite biosynthetic genes coding for non-ribosomal peptide synthetases (NRPSs), and polyketide synthases (PKS). The moonmilk Streptomyces collection was found to display strong inhibitory activities against a wide range of reference organisms, as 94, 71, and 94% of the isolates inhibited or impaired the growth of Gram-positive, Gram-negative bacteria, and fungi, respectively. Interestingly, 90% of the cave strains induced strong growth suppression against the multi-drug resistant Rasamsonia argillacea, a causative agent of invasive mycosis in cystic fibrosis and chronic granulomatous diseases. No correlation was observed between the global antimicrobial activity of an individual strain and the number of NRPS and PKS genes predicted in its genome, suggesting that approaches for awakening cryptic metabolites biosynthesis should be applied to isolates with no antimicrobial phenotype. Overall, our work supports the common belief that moonmilk might effectively treat various infectious diseases thanks to the presence of a highly diverse population of prolific antimicrobial producing Streptomyces, and thus may indeed constitute a promising reservoir of potentially novel active natural compounds.

Long-Term Rasamsonia argillacea Complex Species Colonization Revealed by PCR Amplification of Repetitive DNA Sequences in Cystic Fibrosis Patients.

The aim of this work was to document molecular epidemiology of Rasamsonia argillacea species complex isolates from cystic fibrosis (CF) patients. In this work, 116 isolates belonging to this species complex and collected from 26 CF patients and one patient with chronic granulomatous disease were characterized using PCR amplification assays of repetitive DNA sequences and electrophoretic separation of amplicons (rep-PCR). Data revealed a clustering consistent with molecular species identification. A single species was recovered from most patients. Rasamsonia aegroticola was the most common species, followed by R. argillacea sensu stricto and R. piperina, while R. eburnea was not identified. Of 29 genotypes, 7 were shared by distinct patients while 22 were patient specific. In each clinical sample, most isolates exhibited an identical genotype. Genotyping of isolates recovered from sequential samples from the same patient confirmed the capability of R. aegroticola and R. argillacea isolates to chronically colonize the airways. A unique genotype was recovered from two siblings during a 6-month period. In the other cases, a largely dominant genotype was detected. Present results which support the use of rep-PCR for both identification and genotyping for the R. argillacea species complex provide the first molecular evidence of chronic airway colonization by these fungi in CF patients.

Azole Resistance in Moulds—Approach to Detection in a Clinical Laboratory

The multitude of factors has contributed to the increasing number of fungal infections caused by species of difficult-to-treat opportunistic moulds, such as Fusarium, Scedosporium, and cryptic Aspergilli. Also, rare fungi sporadically encountered, such as Rasamsonia argillacea, Penicillium oxalicum, and melanized fungi, are now well recognized. The high mortality associated with these rare and uncommon fungi is primarily linked to the difficulty in diagnosis and limited therapeutic options, as many of them exhibit resistance to antifungals including azoles. Azole resistance in Aspergillus fumigatus has been increasingly reported because standardized methods for susceptibility testing and associated clinical breakpoints and epidemiological cutoff values became available. However, such advances in antifungal susceptibility testing (AFST) in non-Aspergillus moulds barring mucorales have been lacking. Notwithstanding the fact that the true incidence of these non-Aspergillus filamentous moulds in clinical settings is hitherto unknown, also data on AFST by standardized methods is largely lacking. Determination of minimum inhibitory concentration (MIC) by reference techniques is the gold standard to detect azole resistance in filamentous fungi. In recent years, some progress has been made toward the description of resistance mechanisms at molecular level especially in Aspergillus. This paper reviews the present state of azole resistance in Aspergillus and other filamentous mould species and discusses their relevance to clinical practice.

56 Colonization by Rasamsonia argillacea in cystic fibrosis patients: A two-year retrospective study

Objective Rasamsonia is a new genus which comprises both thermotolerant Talaromyces and thermophilic Geosmithia species. Rasamsonia argillacea (RA) has recently been reported as the cause of invasive mycosis in human chronic granulomatous disease and also as airway colonizer in patients with cystic fibrosis (CF). The aim of this work is to follow patients colonized by emerging fungus Rasamsonia and study their clinical characteristics and conditions. Methods Over a two years period (2013–2014), seven out of the 798 CF patients, followed at Milan centre, showed at least one positive culture for Rasamsonia . RA was identified macroscopically and microscopically by lactic blue stain. Molecular biology analysis on RA strains will be carried out. Results No correlation between RA colonization and patient sex (4F, 3M) was found. Six patients carried at least one copy of mutated allele DF508. Five patients received antimycotic oral therapy (4 itraconazole and 1 voriconazole) before RA recovery. Two out of the patients (both treated with itraconazole) were intermittently colonized with RA. One of patients was also chronically colonized with Scedosporium apiospermum. Pulmonary function (FEV1) remained substantially unvaried. Conclusion All patients were chronically colonized also by various bacteria species: probably the presence of bacteria and the resulting inflammatory response can predispose CF airways to RA acquisition. Further studies are needed to asses the possible association between genotype of patients and colonization by RA, whether antimycotic administration could be a risk factor for RA acquisition and to investigate the RA impact on pulmonary function.

The Airway Colonization by Opportunistic Filamentous Fungi in Patients with Cystic Fibrosis: Recent Updates

There has been a remarkable increase recently in the isolation of fungi such as Aspergillus fumigatus, species of the Pseudallescheria boydii/Scedosporium apiospermum complex, and Exophiala sp. from the respiratory tract of patients with cystic fibrosis (CF). This review describes the recent insights into the epidemiology, ecology, and physiopathology of the filamentous fungi able to colonize and/or to infect the airways of CF patients, and that may be responsible for accelerated lung function decline. We summarize salient features not only on highly prevalent species such as Aspergillus and Scedosporium, but also on more recently described fungi such as Rasamsonia argillacea. In addition, we discuss the challenges inherent in tracking and interpreting rates of fungal colonization/infection in the CF patient population, taking into consideration the polymicrobial composition of the CF lung environment, the corresponding cross-kingdom interactions, and the new concept of mycobiota. Further research is warranted to clarify the role of fungi in CF lung disease and its therapeutic management.

Validation of a novel real-time PCR for detecting Rasamsonia argillacea species complex in respiratory secretions from cystic fibrosis patients.

Members of the recently introduced fungal genus Rasamsonia (formerly included in the Geosmithia genus) have been described as emerging pathogens in immunosuppressed hosts or patients with cystic fibrosis (CF). Rasamsonia species have often been misidentified as Penicillium or Paecilomyces because of similar morphological characteristics. We validated a commercially available real-time PCR assay (Primerdesign™, UK) for accurate detection of species from the Rasamsonia argillacea complex. First, we tested this assay with a collection of 74 reference strains and clinical isolates and then compared the PCR with cultures of 234 respiratory samples from 152 patients with CF from two University Hospitals in Germany and France. The assay reliably detected the three main species within the Rasamsonia argillacea species complex (R. argillacea, R. piperina, R. aegroticola), which are typically encountered in CF patients. The limit of DNA detection was between 0.01 and 1 pg/μL. Analysis of the DNA extracts from respiratory specimens of CF patients revealed that four out of the 153 patients studied (2.6%) were colonized with R. argillacea species complex. Two species from the R. argillacea complex grew in the parallel cultures from the same patients. In one patient the PCR was positive 5 months before culture. The real-time PCR assay is a sensitive and specific method for detecting the three most important species of the R. argillacea species complex encountered in the CF context. Detection of these emerging pathogens in respiratory secretions from CF patients by this novel assay may increase our understanding of the occurrence and epidemiology of the R. argillacea species complex.

Complexities associated with the molecular and proteomic identification of Paecilomyces species in the clinical mycology laboratory

Paecilomyces species are emerging fungal pathogens. Morphological identifications are complicated by similarities among the members of the P. variotii complex as well as to some Rasamsonia and Hamigera species. The purpose of this study was to compare matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) with molecular diagnostic standards (i.e., multilocus DNA sequencing of the internal transcribed spacer regions 1 and 2, D1/D2 regions, and part of the β-tubulin gene) for the identification of Paecilomyces spp. encountered in two clinical mycology laboratories. A total of 77 clinical isolates identified morphologically as P. variotii (n = 21), P. lilacinus (n = 52), and Paecilomyces spp. not otherwise specified (n=4) were included. In accord with the most recent taxonomy, all P. lilacinus isolates were confirmed as Purpureocillium lilacinum by both sequencing and MALDI-TOF MS. Fungi phenotypically resembling P. variotii or Paecilomyces spp. were identified by molecular techniques as P. variotii sensu stricto (n=12), P. formosus (n=3), P. dactylethromorphus (n=3), Rasamsonia argillacea (n=4), or R. piperina (n=1) and at the genus level as an isolate of a Hamigera sp. and a Paecilomyces sp. There was 92.2% (71/77) agreement between the molecular and proteomic methods only after supplementation of the MALDI-TOF MS database with type strains. Paecilomyces variotii-like organisms required multilocus DNA interrogations for differentiation and account for all of the fungi whose identification was missed by MALDI-TOF MS. Overall, MALDI-TOF MS was a rapid and reliable alternative to multilocus sequencing. However, significant augmentation of the commercially available database was required to reproducibly identify this group of important human pathogens.

Rasamsonia Argillacea Species Complex: Taxonomy, Pathogenesis and Clinical Relevance

Since 2010, colonizations/infections by Rasamsonia argillacea species complex, previously known as Geosmithia argillacea, have been regularly reported in literature. We reviewed all available cases focusing on pathogenesis and clinical relevance. The number of cases may be underestimated, as these fungi are frequently misidentified as Penicillium or Paecilomyces species. Major underlying conditions that predispose for infections by the R. argillacea species complex include cystic fibrosis (CF) and chronic granulomatous disease (CGD). While the pathogenic role of the colonization of CF lungs is still under debate, these molds are the causative agent of pneumonia and/or invasive infections in CGD patients. Given their thermotolerance and their resistance to various antifungals, especially the azole drugs, a special attention should be paid to the chronic colonization of the airways by these fungi in CF and CGD patients.