Epidemiological investigation for grouped cases of Trichosporon asahii using whole genome and IGS1 sequencing.

BACKGROUND: Trichosporonosis is a rare invasive infection in humans mainly due to Trichosporon asahii, and especially recovered from patients having haematological malignancy. Since 2012, IGS1 region sequencing is used as a genotyping method to distinguish isolates, with high frequency of one haplotype worldwide and a geographic specificity for some haplotypes. OBJECTIVES: We compared the IGS1 genotyping method and whole genome sequencing (WGS) to study the relationship between clinical isolates involved in two grouped cases in France. METHODS: IGS1 sequencing and antifungal susceptibility testing were performed for 54 clinical isolates. Clinical data for 28 isolates included in surveillance programs were analysed. Whole genome was sequenced for 32 clinical isolates and the type strain. RESULTS: All isolates were intrinsically resistant to flucytosine, while voriconazole had the most potent in vitro activity. The majority of the isolates was recovered from patients with haematological malignancies (42.86%), with a high proportion of children (<15 yrs-old, 32.14%) and a high mortality rate at three months (46.15%). Based on the WGS analysis, isolates exhibiting IGS1 haplotype 1, 3 and 7 belonged to different clades. Five isolates recovered during the first grouped cases had the same IGS1 haplotype and shared 99% of SNPs similarity. For the second grouped cases, four isolates had 98.7% of SNPs similarity while the isolate recovered 4 years earlier was totally unlinked. CONCLUSIONS: We confirmed the usefulness of IGS1 sequencing for grouped cases infection of T. asahii. We underlined its limitation for the study of population structure and the utility of WGS analysis for the study of epidemiologically unrelated isolates.

Toward the Standardization of Mycological Examination of Sputum Samples in Cystic Fibrosis: Results from a French Multicenter Prospective Study.

Fungal respiratory colonization of cystic fibrosis (CF) patients emerges as a new concern; however, the heterogeneity of mycological protocols limits investigations. We first aimed at setting up an efficient standardized protocol for mycological analysis of CF sputa that was assessed during a prospective, multicenter study: "MucoFong" program (PHRC-06/1902). Sputa from 243 CF patients from seven centers in France were collected over a 15-month period and submitted to a standardized protocol based on 6 semi-selective media. After mucolytic pretreatment, sputa were plated in parallel on cycloheximide-enriched (ACT37), erythritol-enriched (ERY37), benomyl dichloran-rose bengal (BENO37) and chromogenic (CAN37) media incubated at 37 °C and on Sabouraud-chloramphenicol (SAB27) and erythritol-enriched (ERY27) media incubated at 20-27 °C. Each plate was checked twice a week during 3 weeks. Fungi were conventionally identified; time for detection of fungal growth was noted for each species. Fungal prevalences and media performances were assessed; an optimal combination of media was determined using the Chi-squared automatic interaction detector method. At least one fungal species was isolated from 81% of sputa. Candida albicans was the most prevalent species (58.8%), followed by Aspergillus fumigatus (35.4%). Cultivation on CAN37, SAB27, ACT37 and ERY27 during 16 days provided an optimal combination, detecting C. albicans, A. fumigatus, Scedosporium apiospermum complex and Exophiala spp. with sensitivities of 96.5, 98.8, 100 and 100%. Combination of these four culture media is recommended to ensure the growth of key fungal pathogens in CF respiratory specimens. The use of such consensual protocol is of major interest for merging results from future epidemiological studies.

Reappraisal of Conventional Diagnosis for Dermatophytes.

Dermatophytoses include a wide variety of diseases involving glabrous skin, nails and hair. These superficial infections are a common cause of consultation in dermatology. In many cases, their diagnosis is not clinically obvious, and mycological analysis therefore is required. Direct microscopic examination of the samples using clearing agents provides a quick response to the clinician and is usually combined with cultures on specific media, which must be used to overcome the growth of contaminating moulds that may hamper the recovery of dermatophytes. Accurate identification of the causative agent (i.e. at the species level), currently based on morphological criteria, is necessary not only to initiate an appropriate treatment but also for setting prophylactic measures. However, conventional methods often lack sensitivity and species identification may require up to 4 weeks if subcultures are needed. Histological analysis, which is considered the "gold standard" for the diagnosis of onychomycoses, is seldom performed, and as direct examination, it does not allow precise identification of the pathogen. Nevertheless, a particular attention to the quality of clinical specimens is warranted. Moreover, the sensitivity of direct examination may be greatly enhanced by the use of fluorochromes such as calcofluor white. Likewise, sensitivity of the cultures could be enhanced by the use of culture media containing antifungal deactivators. With the generalization of molecular identification by gene sequencing or MALDI-TOF mass spectrometry, the contribution of historical biochemical or physiological tests to species identification of atypical isolates is now limited. Nevertheless, despite the recent availability of several PCR-based kits and an extensive literature on molecular methods allowing the detection of fungal DNA or both detection and direct identification of the main dermatophyte species, the biological diagnosis of dermatophytosis in 2016 still relies on both direct examination and cultures of appropriate clinical specimens.

Self-assembly of proteins into a three-dimensional multilayer system: investigation of the surface of the human fungal pathogen Aspergillus fumigatus.

Hydrophobins are small surface active proteins that fulfil a wide spectrum of functions in fungal growth and development. The human fungal pathogen Aspergillus fumigatus expresses RodA hydrophobins that self-assemble on the outer conidial surface into tightly organized nanorods known as rodlets. AFM investigation of the conidial surface allows us to evidence that RodA hydrophobins self-assemble into rodlets through bilayers. Within bilayers, hydrophilic domains of hydrophobins point inward, thus making a hydrophilic core, while hydrophobic domains point outward. AFM measurements reveal that several rodlet bilayers are present on the conidial surface thus showing that proteins self-assemble into a complex three-dimensional multilayer system. The self-assembly of RodA hydrophobins into rodlets results from attractive interactions between stacked ß-sheets, which conduct to a final linear cross-ß spine structure. A Monte Carlo simulation shows that anisotropic interactions are the main driving forces leading the hydrophobins to self-assemble into parallel rodlets, which are further structured in nanodomains. Taken together, these findings allow us to propose a mechanism, which conducts RodA hydrophobins to a highly ordered rodlet structure. The mechanism of hydrophobin assembly into rodlets offers new prospects for the development of more efficient strategies leading to disruption of rodlet formation allowing a rapid detection of the fungus by the immune system.

Active Surveillance Program to Increase Awareness on Invasive Fungal Diseases: the French RESSIF Network (2012 to 2018).

The French National Reference Center for Invasive Mycoses and Antifungals leads an active and sustained nationwide surveillance program on probable and proven invasive fungal diseases (IFDs) to determine their epidemiology in France. Between 2012 and 2018, a total of 10,886 IFDs were recorded. The incidence increased slightly over time (2.16 to 2.36/10,000 hospitalization days, P = 0.0562) in relation with an increase of fungemia incidence (1.03 to 1.19/10,000, P = 0.0023), while that of other IFDs remained stable. The proportion of ≥65-year-old patients increased from 38.4% to 45.3% (P < 0.0001). Yeast fungemia (n = 5,444) was due mainly to Candida albicans (55.6%) with stable proportions of species over time. Echinocandins became the main drug prescribed (46.7% to 61.8%), but global mortality rate remained unchanged (36.3% at 1 month). Pneumocystis jirovecii pneumonia (n = 2,106) was diagnosed mostly in HIV-negative patients (80.7%) with a significantly higher mortality than in HIV-positive patients (21.9% versus 5.4% at 1 month, P < 0.0001). Invasive aspergillosis (n = 1,661) and mucormycosis (n = 314) were diagnosed mostly in hematology (>60% of the cases) with a global mortality rate of 42.5% and 59.3%, respectively, at 3 months and significant changes in diagnosis procedure over time. More concurrent infections were also diagnosed over time (from 5.4% to 9.4% for mold IFDs, P = 0.0115). In conclusion, we observed an aging of patients with IFD with a significant increase in incidence only for yeast fungemia, a trend toward more concurrent infections, which raises diagnostic and therapeutic issues. Overall, global survival associated with IFDs has not improved despite updated guidelines and new diagnostic tools. IMPORTANCE The epidemiology of invasive fungal diseases (IFDs) is hard to delineate given the difficulties in ascertaining the diagnosis that is often based on the confrontation of clinical and microbiological criteria. The present report underlines the interest of active surveillance involving mycologists and clinicians to describe the global incidence and that of the main IFDs. Globally, although the incidence of Pneumocystis pneumonia, invasive aspergillosis, and mucormycosis remained stable over the study period (2012 to 2018), that of yeast fungemia increased slightly. We also show here that IFDs seem to affect older people more frequently. The most worrisome observation is the lack of improvement in the global survival rate associated with IFDs despite the increasing use of more sensitive diagnostic tools, the availability of new antifungal drugs very active in clinical trials, and a still low/marginal rate of acquired in vitro resistance in France. Therefore, other tracks of improvement should be investigated actively.

Geosmithia argillacea: an emerging pathogen in patients with cystic fibrosis.

We report eight cases of airway colonization by Geosmithia argillacea in patients with cystic fibrosis. This filamentous fungus, resembling members of the genera Penicillium and Paecilomyces, was identified by molecular analysis. All patients carried a mutation on each CFTR (cystic fibrosis transmembrane conductance regulator) allele, with at least one copy of the F508del mutation. The first isolation of this fungus occurred from F508del-homozygous patients at a younger age than in F508del-heterozygous patients. Before recovery of G. argillacea, all patients were treated with itraconazole; two of them had also received voriconazole for an Aspergillus fumigatus infection. However, antifungal susceptibility patterns showed high MICs of voriconazole for all isolates, and high MICs of amphotericin B and itraconazole for the majority of them, but mostly low minimum effective concentrations (MECs) of caspofungin. The appearance and persistence of G. argillacea in the airways were not associated with exacerbation of the disease. However, the clinical implications of G. argillacea, particularly in immunocompromised patients, remain a concern, particularly given recent observations suggesting that this fungus may also cause disseminated infections.

Unusual Aspergillus species in patients with cystic fibrosis.

Poorly sporulating Aspergillus isolates from patients with cystic fibrosis (CF) are generally identified in routine procedures as Aspergillus spp. In this study, we identified and characterized 11 isolates belonging to two unusual Aspergillus species of the section Fumigati (A. lentulus and Neosartorya pseudofischeri) recovered from four different patients. Aspergillus lentulus was found occasionally during a 10-year follow-up study of one CF patient colonized by A. fumigatus. Neosartorya pseudofischeri was isolated from three patients followed in different European hospitals. This species was recovered from two sputum samples of one patient, and from four successive samples of the two other patients, suggesting that it may be responsible for chronic colonization. Both species were isolated together with A. fumigatus. Isolates from both species did not grow at 50°C, and DNA sequence analysis, together with further morphological observations permitted identification at the species level. Growth at different temperatures and antifungal susceptibility were also investigated. All the isolates of N. pseudofischeri exhibited a very low susceptibility to voriconazole (VRZ) whereas a very low susceptibility to VRZ and amphotericin B was seen with the A. lentulus isolates.

Acremonium strictum fungaemia in a paediatric immunocompromised patient: diagnosis and treatment difficulties.

During the past two decades, an increasing number of unusual moulds has been reported as responsible for septicaemia and systemic or disseminated infections in immunocompromised patients. Investigation of fever in a 10-year-old boy with acute myeloblastic leukaemia, including blood cultures on selective media, allowed the diagnosis of a fungaemia due to the slow-growing fungus Acremonium strictum. The patient recovered with liposomal amphotericin B (AmB) and voriconazole, followed by voriconazole alone due to AmB resistance. Facing a neutropenic patient with fever, clinicians usually suspect bacterial or viral aetiologies. This case, however, illustrates the need for mycological analysis of blood samples in febrile neutropenic patients and for antifungal susceptibility testing.

Development of an oligonucleotide array for direct detection of fungi in sputum samples from patients with cystic fibrosis.

Cystic fibrosis (CF) is the most common inherited genetic disease in Caucasian populations. Besides bacteria, many species of fungi may colonize the respiratory tract of these patients, sometimes leading to true respiratory infections. In this study, an oligonucleotide array capable of identifying 20 fungal species was developed to directly detect fungi in the sputum samples of CF patients. Species-specific oligonucleotide probes were designed from the internal transcribed spacer (ITS) regions of the rRNA operon and immobilized on a nylon membrane. The fungal ITS regions were amplified by PCR and hybridized to the array for species identification. The array was validated by testing 182 target strains (strains which we aimed to identify) and 141 nontarget strains (135 species), and a sensitivity of 100% and a specificity of 99.2% were obtained. The validated array was then used for direct detection of fungi in 57 sputum samples from 39 CF patients, and the results were compared to those obtained by culture. For 16 sputum samples, the results obtained by the array corresponded with those obtained by culture. For 33 samples, the array detected more fungal species than culture did, while the reverse was found for eight samples. The accuracy of the array for fungal detection in sputum samples was confirmed (or partially confirmed) in some samples by cloning and resequencing the amplified ITS fragments. The present array is a useful tool for both the simultaneous detection of multiple fungal species present in the sputa of CF patients and the identification of fungi isolated from these patients.

Melanin is an essential component for the integrity of the cell wall of Aspergillus fumigatus conidia.

BACKGROUND: Aspergillus fumigatus is the most common agent of invasive aspergillosis, a feared complication in severely immunocompromised patients. Despite the recent commercialisation of new antifungal drugs, the prognosis for this infection remains uncertain. Thus, there is a real need to discover new targets for therapy. Particular attention has been paid to the biochemical composition and organisation of the fungal cell wall, because it mediates the host-fungus interplay. Conidia, which are responsible for infections, have melanin as one of the cell wall components. Melanin has been established as an important virulence factor, protecting the fungus against the host's immune defences. We suggested that it might also have an indirect role in virulence, because it is required for correct assembly of the cell wall layers of the conidia. RESULTS: We used three A. fumigatus isolates which grew as white or brown powdery colonies, to demonstrate the role of melanin. Firstly, sequencing the genes responsible for biosynthesis of melanin (ALB1, AYG1, ARP1, ARP2, ABR1 and ABR2) showed point mutations (missense mutation, deletion or insertion) in the ALB1 gene for pigmentless isolates or in ARP2 for the brownish isolate. The isolates were then shown by scanning electron microscopy to produce numerous, typical conidial heads, except that the conidia were smooth-walled, as previously observed for laboratory mutants with mutations in the PKSP/ALB1 gene. Flow cytometry showed an increase in the fibronectin binding capacity of conidia from mutant isolates, together with a marked decrease in the binding of laminin to the conidial surface. A marked decrease in the electronegative charge of the conidia and cell surface hydrophobicity was also seen by microelectrophoresis and two-phase partitioning, respectively. Ultrastructural studies of mutant isolates detected considerable changes in the organisation of the conidial wall, with the loss of the outermost electron dense layer responsible for the ornamentations seen on the conidial surface in wild-type strains. Finally, analysis of the conidial surface of mutant isolates by atomic force microscopy demonstrated the absence of the outer cell wall rodlet layer which is composed of hydrophobins. CONCLUSION: These results suggest that, in addition to a protective role against the host's immune defences, melanin is also a structural component of the conidial wall that is required for correct assembly of the cell wall layers and the expression at the conidial surface of adhesins and other virulence factors.

Occurrence and relevance of filamentous fungi in respiratory secretions of patients with cystic fibrosis--a review.

The colonization of airways by filamentous fungi and the development of respiratory infections require some predisposing factors as encountered in patients with cystic fibrosis (CF). Indeed, the defective mucociliary clearance which characterizes the disease is associated with local immunological disorders. In addition, the prolonged therapy with antibiotics and the use of corticosteroid treatments also facilitate fungal growth. An important fungal biota has been described in respiratory secretions of patients suffering from CF. Aspergillus fumigatus, Scedosporium apiospermum and Aspergillus terreus for filamentous fungi and Candida albicans for yeasts are the main fungal species associated with CF. Although less common, several fungal species including Aspergillus flavus and Aspergillus nidulans may be isolated transiently from CF respiratory secretions, while others such as Exophiala dermatitidis and Scedosporium prolificans may chronically colonize the airways. Moreover, some of them like Penicillium emersonii and Acrophialophora fusispora are encountered in humans almost exclusively in the context of CF. As fungal complications in CF patients are essentially caused by filamentous fungi the present review will not include works related to yeasts. In CF patients, fungi may sometimes be responsible for deterioration of lung function, as occurs in allergic broncho-pulmonary aspergillosis (ABPA) which is the most common fungal disease in this context. Additionally, although the clinical relevance of the fungal airway colonization is still a matter of debate, filamentous fungi may contribute to the local inflammatory response, and therefore to the progressive deterioration of the lung function.

Isolation of Trichophyton concentricum from chronic cutaneous lesions in patients from the Solomon Islands.

Tinea imbricata, also known as 'Tokelau', is an uncommon superficial mycosis caused by the anthropophilic dermatophyte Trichophyton concentricum. Cutaneous lesions appear characteristically as scaly and concentric rings that may cover all parts of the body. Often acquired in childhood, tinea imbricata is a chronic disease and lichenification is extremely common due to pruritus. The dermatophytosis mainly occurs in the South Pacific, but also in some regions of Southeast Asia and Central or South America. Tinea imbricata usually affects people living in primitive and isolated conditions. Mycological analysis is required for the diagnosis. The epidemiological and mycological study reported here took place in the Solomon Islands from June-September 2006. Skin scrapings were collected from 29 Melanesian patients (aged 8 months to 58 years) with chronic cutaneous lesions and were analysed mycologically in the Laboratory of Parasitology and Mycology of Angers University Hospital (France). Ten patients showed very evocative lesions with a positive direct examination, but T. concentricum was only isolated from three patients. Identification of the strains was confirmed by sequencing of the internal transcribed spacer (ITS) regions. With the increase in international travel, one cannot disregard that this very rare species may be isolated by mycologists in temperate areas from patients coming from endemic foci.

Early Detection of Schistosoma Egg-Induced Pulmonary Granulomas in a Returning Traveler.

We report the case of a French traveler who developed acute pulmonary schistosomiasis 2 months after visiting Benin. He presented with a 1-month history of fever, cough, and thoracic pain. Initial investigations revealed hypereosinophilia and multiple nodular lesions on chest computed tomography scan. Lung biopsies were performed 2 months later because of migrating chest infiltrates and increasing eosinophilia. Histological examination showed schistosomal egg-induced pulmonary granulomas with ova exhibiting a prominent terminal spine, resembling Schistosoma haematobium. However, egg shells were Ziehl-Neelsen positive, raising the possibility of a Schistosoma intercalatum or a Schistosoma guineensis infection. Moreover, involvement of highly infectious hybrid species cannot be excluded considering the atypical early pulmonary oviposition. This case is remarkable because of the rarity of pulmonary schistosomiasis, its peculiar clinical presentation and difficulties in making species identification. It also emphasizes the need to consider schistosomiasis diagnosis in all potentially exposed travelers with compatible symptoms.

Diagnosis of dermatophytosis: an evaluation of direct examination using MycetColor® and MycetFluo®.

Dermatophytes are an important cause of superficial fungal infection. Direct examination of skin, nail, or hair samples remains essential in diagnosis, as it provides a quick response to the clinician. However, mycological analysis, including direct examination and culture, often lacks sensitivity. The use of stains or fluorochromes may enhance the performance of direct examination. We analyzed 102 samples from patients with suspected dermatophytosis in 4 different diagnostic mycology laboratories. Two reagents, MycetColor® and MycetFluo®, which use Congo red and calcofluor dye, respectively, were evaluated for the direct microscopic examination of skin, hair, and nail specimens. The results were compared to those of culture and conventional direct examination. Both reagents were able to clarify the specimens and also to specifically stain fungal elements. Microscopic examination of the specimens was greatly facilitated with MycetFluo®, which allowed a higher number of positive cases to be detected compared to the other methods.

Is procalcitonin increased in cases of invasive amoebiasis? A retrospective, observational study.

Procalcitonin (PCT) levels are commonly used for diagnostic guidance in routine bacterial infections. By contrast, little data are currently available regarding PCT in parasitic diseases, and its role in cases of invasive amoebiasis has not yet been described. For this purpose, 35 adult patients with a proven diagnosis of invasive or digestive amoebiasis were included in a 4-year study period. Serum PCT was retrospectively assessed. Results were analysed with regard to the usual inflammatory biomarkers, like C-reactive protein (CRP). PCT was significantly higher in patients with proven invasive amoebiasis than in digestive amoebiasis (mean value: 4.03 µg/L versus 0.07 µg/L, respectively; P < 0.001), but the SD was greater than with CRP, and the effect was less than that demonstrated in bacterial infections. By contrast, PCT was not shown to be elevated during digestive amoebiasis.

Laser nephelometry applied in an automated microplate system to study filamentous fungus growth.

By contrast with photometry (i.e., the measurement of light transmitted through a particle suspension), nephelometry is a direct method of measuring light scattered by particles in suspension. Since the scattered light intensity is directly proportional to the suspended particle concentration, nephelometry is a promising method for recording microbial growth and especially for studying filamentous fungi, which cannot be efficiently investigated through spectrophotometric assays. We describe herein for the first time a filamentous fungi-tailored procedure based on microscale liquid cultivation and automated nephelometric recording of growth, followed by extraction of relevant variables (lag time and growth rate) from the obtained growth curves. This microplate reader technique is applicable for the evaluation of antifungal activity and for large-scale phenotypic profiling.

Conventional methods for the diagnosis of dermatophytosis.

Dermatophytes are keratinolytic fungi responsible for a large variety of diseases that can affect glabrous skin, nails and hair. In many cases, the diagnosis is not clinically obvious, and mycological analysis is required. This includes both direct microscopic examination and cultures. First of all, clinical specimens have to be sampled according to localization and characteristics of the lesions. Direct microscopic examination is usually performed using clearing reagents (KOH or Amman's chloral-lactophenol), but its sensitivity may be greatly enhanced by the use of stains or fluorochromes such as Congo red or Calcofluor white. Histological analysis is an efficient method, but it is constraining for the patients and, as direct examination, it does not allow precise identification of the pathogen. Cultures are therefore needed, and specific culture media may be used to overcome the growth of rapidly growing contaminating moulds which may hamper the recovery of dermatophytes. Identification at the species level which may be useful to initiate an appropriate treatment or for setting prophylactic measures, relies on macroscopic and microscopic morphology. Subcultures on culture media which stimulate conidiation and, for some species, the production of pigments, are often necessary. Additionally, in case of atypical isolates, some biochemical or physiological tests may be performed such as the search for urease activity or the in vitro hair perforation test. However, their contribution to species identification is rather limited, and progress is still needed for the development of biochemical or immunological tests allowing an accurate identification at the species level, pending for the availability of molecular biology-based kits.

Adherence mechanisms in human pathogenic fungi.

Interactions of human pathogenic fungi with the host tissues are key factors in the pathogenesis of mycoses. Based on the concept that adherence of microorganisms is a prerequisite for initiation of the disease, numerous studies have been conducted to identify the fungal adhesins and their respective receptors. Several adhesins recognizing different host ligands, sometimes with multifunctional properties, have been described. Some of them have been extensively characterized, and their expression analyzed according to morphological changes or culture conditions. For some ligands, the amino acid or carbohydrate motifs participating in these interactions have been identified. Various host proteins or glycoproteins have been suggested as ligands, including components of biological fluids, or extracellular matrix and basement membrane proteins; equally adherence to several cell types, mainly epithelial and endothelial cells, or to biomaterials has been considered. This review synthesizes available information regarding adherence of the most important human fungal pathogens. It is divided into three sections corresponding to the three main groups of pathogenic fungi: Candida yeasts, opportunistic moulds and other filamentous fungal pathogens, and dimorphic fungi.