Chronic mucocutaneous candidiasis : a model for studying genomic adaptation in Candida albicans

Candida albicans est une levure commensale du tube digestif de l’Homme mais également un pathogène opportuniste responsable d’infections dont la gravité est fonction des défenses immunitaires de l’hôte. Au cours de l’interaction avec l’hôte, cette levure est confrontée à de nombreux stress environnementaux et immunitaires imposant des capacités d’adaptation rapides pour survivre. Le travail de thèse présenté dans ce manuscrit est consacré à l'étude de la diversité génomique de C. albicans chez l’individu sain mais également au cours de l’interaction pathologique de longue durée chez l’Homme. Dans une première partie nous montrons qu’il existe un niveau élevé d’hétérogénéité génomique essentiellement dus à des évènements de perte d’hétérozygoties, entre les isolats de C. albicans issus de prélèvements buccaux de porteurs sains et démontrons que celle-ci n’est pas le reflet de problèmes inhérents à la technique et à l’analyse des données de séquençage à haut débit. Puis, nous avons évalué dans quelle mesure le génome de C. albicans était capable d’évoluer, dans un contexte pathologique de longue durée grâce à l’étude de souches chronologiques provenant de patients atteints de candidose cutanéo-muqueuse chronique. Nous montrons qu’il existe une importante dynamique dans l’apparition et l’élimination de variants phénotypiques et génotypique au cours de l’interaction pathologique chez l’hôte pouvant être la conséquence de l'adaptation des souches à l’interaction pathologique chronique. Un phénotype d’importance est la survenue de résistance de ces souches aux antifongiques. Une étude approfondie des gènes impliqués dans la résistance aux antifongiques a permis de décrire de nouvelles mutations dans les gènes ERG11 et TAC1 impliquées dans la résistance aux antifongiques azolés.Candida albicans is a common component of the human digestive tract and is considered the major opportunistic fungal pathogen. During interaction with the host, this yeast is confronted with numerous environmental and immune stresses imposing rapid adaptation capacities to survive. This manuscript aimed to study the genomic diversity of C. albicans, in healthy individuals and during long-term pathological interaction. In the first part of this work, we showed that there is a high level of genomic heterogeneity, especially linked to loss-of-heterozygosity, between isolates of C. albicans in oral samples from healthy carriers. Furthermore, we demonstrated that the heterogeneity observed is not reflective of technical problems nor associated to the high throughput sequencing data analysis. Then, we evaluated to which extent the C. albicans genome was able to evolve in a long-term pathological context, thanks to the study of chronological strains isolated from patients suffering from chronic mucocutaneous candidiasis. We have shown that there is an important dynamic in the appearance and the elimination of phenotypic and genotypic variants during the pathological interaction with the host. This may be the consequence of the adaptation of the strains to the chronic pathological interaction with its host. One of the interesting phenotype is the appearance of resistance to antifungal agents in these strains. An in-depth study of the genes involved in resistance to antifungal agents has enabled the detection and the description of new mutations in the ERG11 and TAC1 genes, involved in resistance to azole antifungals

Candida albicans and Candida dubliniensis Show Different Trailing Effect Patterns When Exposed to Echinocandins and Azoles

International audienceWhen Candida albicans and Candida dubliniensis isolates were tested for susceptibility to fluconazole and echinocandins using either EUCAST or Etest methods, differential patterns of growth were observed, independently of the methods used. For C. albicans, a trailing phenomenon (incomplete growth inhibition at supra-MICs) was observed with fluconazole in 90% and 93.3% for EUCAST and Etest, respectively, but not with echinocandins (50% for EUCAST and >86% for Etest). This suggests that the pathways involved in the trailing effect might be different between these two related species. Furthermore, clinical microbiologists must be aware of these species-specific patterns for a reliable MIC determination

Within-host genomic diversity of candida albicans in healthy carriers

International audienceGenomic variations in Candida albicans, a major fungal pathogen of humans, have been observed upon exposure of this yeast to different stresses and experimental infections, possibly contributing to subsequent adaptation to these stress conditions. Yet, little is known about the extent of genomic diversity that is associated with commensalism, the predominant lifestyle of C. albicans in humans. In this study, we investigated the genetic diversity of C. albicans oral isolates recovered from healthy individuals, using multilocus sequencing typing (MLST) and whole genome sequencing. While MLST revealed occasional differences between isolates collected from a single individual, genome sequencing showed that they differed by numerous single nucleotide polymorphisms, mostly resulting from short-range loss-of-heterozygosity events. These differences were shown to have occurred upon human carriage of C. albicans rather than subsequent in vitro manipulation of the isolates. Thus, C. albicans intra-sample diversity appears common in healthy individuals, higher than that observed using MLST. We propose that diversifying lineages coexist in a single human individual, and this diversity can enable rapid adaptation under stress exposure. These results are crucial for the interpretation of longitudinal studies evaluating the evolution of the C. albicans genome

Azole preexposure affects the Aspergillus fumigatus population in patients

International audienceThe relationship between the azole preexposure of 86 patients and the genotype, azole susceptibility, and cyp51A polymorphisms of 110 corresponding Aspergillus fumigatus isolates was explored. Isolates carrying serial polymorphisms (F46Y and M172V with or without N248T with or without D255E with or without E427K) had higher itraconazole MICs (P = 0.04), although \textless2 μg/ml using the EUCAST methodology, were associated with two genetic clusters (P \textless 0.001) and with voriconazole preexposure of patients (P = 0.016). Voriconazole preexposure influences the distribution of A. fumigatus isolates with selection of isolates carrying cyp51A polymorphisms and higher itraconazole MICs

Matrix-assisted laser desorption ionization-time of flight mass spectrometry for fast and accurate identification of Pseudallescheria/Scedosporium species

International audienceCurrent identification of Scedosporium spp. based on morphological characteristics fails todetect emerging species described using multi-locus sequencing (MLS). Among the 8 recently described phylogenetic species which grouped in five different clades, only Scedosporium apiospermum (clade 4) and Pseudallescheria boydii (clade 5) are known as common pathogens in humans, while the 6 others are emerging, including for exemple Scedosporium aurantiacum (clade 1), Pseudallescheria minutispora (clade 2), or Scedosporium dehoogii (clade 3). In addition, another Scedosporium species, Scedosporium prolificans, which is also common human pathogen but is not included in these clades, cannot be differentiated from those belonging to the above-mentioned clades using morphological techniques. However, accurate identification is important because antifungal drug susceptibility patterns may vary with the species. Matrix-assisted laser desorption ionization-time mass spectrometry (MALDI-TOF MS), allows rapid and reliable identification of microorganisms. We postulated that this method, can also discriminate species of Scedosporium just as we showed for Aspergillus species A set of 7 reference strains belonging to 5 clinically relevant species (S. apiospermum, P. boydii, S. aurantiacum, S. prolificans, P. minutispora) was used to build a reference database Profiles from each referenced strain at different age of the cultures (Days 3, 5, 7) were analyzed to identify species specific discriminating peaks. The spectra of 50 strains (25 P. boydii, 19 S. apiospermum, 3 S. aurantiacum, 2 P.minutispora, 1 S. prolificans) previously identified with MLS (tubulin and/or ITS) were compared to that of each of the reference strains. This database allowed correct identification of 48/50 strains (96%), with no misidentification. Our results, obtained using a simple protocol with no extraction step showed that MALDI-TOF-MS is a powerful tool for rapid identification of clinically relevant species of Scedosporium, including those emerging that cannot currently be identified by micoscopic examination.</p

Human-impacted areas of France are environmental reservoirs of the Pseudallescheria boydii/Scedosporium apiospermum species complex.

International audience: Species of the Pseudallescheria boydii/Scedosporium apiospermum complex (PSC) are emerging fungal pathogens able to chronically colonize the airways of patients with cystic fibrosis (CF). As P. boydii was found more frequently colonizing the lungs of CF patients in France than in other European countries in a previous report, the present study was conducted in order to clarify distribution of PSC species in France and to characterize their natural habitat. The highest densities of PSC isolates were found in human-impacted areas, i.e. agricultural areas, fluids obtained from wastewater treatment plants, playgrounds and industrial areas. PSC was not detected from soil samples collected in forests. Most PSC culture-positive soil samples exhibited a pH range of 6-8. Scedosporium dehoogii, the most abundant species, was detected in all human-impacted area types except vineyards, whereas Scedosporium aurantiacum was mostly found in agricultural areas. Pseudallescheria boydii and S. apiospermum were predominantly isolated from seashores and playgrounds respectively. Pseudallescheria minutispora was found only once from a playground. This study highlights potential sources of contamination of the patients, especially in the CF context

Acquired Flucytosine Resistance during Combination Therapy with Caspofungin and Flucytosine for Candida glabrata Cystitis

International audienceTreatment of Candida glabrata cystitis remains a therapeutic challenge, and an antifungal combination using flucytosine is one option. We describe two patients with refractory C. glabrata cystitis who failed flucytosine combined with caspofungin with early-acquired high-level resistance to flucytosine due to nonsense mutations in the FUR1 gene. Rapidly acquired flucytosine resistance with microbiological failure should discourage combination of caspofungin and flucytosine during urinary candidiasis

Agreement between two real-time commercial PCR kits and an in-house real-time PCR for diagnosis of mucormycosis

ABSTRACT Mucormycosis is a severe and emerging invasive fungal infection associated with high mortality rates. Early diagnosis is crucial for initiating specific antifungal treatment, with molecular tools currently representing the most efficient diagnostic approach. Presently, a standardized in-house real-time PCR method is widely employed for diagnosing mucormycosis. Our study aimed to evaluate the agreement for the Mucorales DNA detection between two commercial real-time PCR assays—the Fungiplex Mucorales Real-Time PCR Kit and the MycoGENIE Aspergillus-Mucorales spp. Real-Time PCR Kit—in comparison with the in-house PCR. We retrospectively analyzed 58 samples previously identified as positive for Mucorales using the in-house PCR. These samples, obtained from 22 patients with proven or probable mucormycosis, were tested with both commercial kits. Additionally, samples from 40 patients without mucormycosis served as negative controls. Our findings revealed that the MycoGENIE Kit demonstrated superior performance in detecting Mucorales DNA in samples identified as positive by the in-house PCR. Notably, we observed minimal variability in cycle threshold (CT) values when comparing the results of the MycoGENIE Kit with those of the in-house PCR, with an average difference of 1.8 cycles. In contrast, the Fungiplex Kit exhibited a larger discrepancy in CT values compared to the in-house PCR, with an average difference of 4.1 cycles. The MycoGENIE Kit exhibited very good agreement (kappa of 0.82) with the in-house PCR for detecting Mucorales DNA across various sample types. These findings are important for the choice of kits that could be used to diagnose mucormycosis in clinical microbiology laboratories.IMPORTANCEEarly diagnosis of mucormycosis is crucial for initiating effective treatment. The detection of Mucorales DNA by PCR in serum has revolutionized the diagnosis of this infection. However, the use of in-house methods can be time consuming. The availability of a commercial kit eliminates the need for in-house assay development, reducing laboratory workload and ensuring consistent performance across different healthcare settings. Currently, there are several commercial assays available, but many have limited evaluation. In this study, we compared two commercial kits and found that the MycoGENIE Kit offers a promising alternative to the in-house method

Evaluation des tests de PCR en temps reel Bio-Evolution Microsporidia generic et typing pour le diagnostic de la microsporidiose intestinale.

International audienceCases of intestinal microsporidiosis infection are underestimated and affect both immunocompromized and immunocompetent patients. Real-time PCR is superseding microscopic examination for its diagnosis in medical analysis laboratories. However, few manufacturers include microsporidia in their PCR panel for the diagnosis of infectious gastroenteritis. Here, we evaluated the performances of the real-time PCR assays microsporidia generic and microsporidia typing (Bio-Evolution, France) on the Rotor-Gene Q real-time PCR cycler (Qiagen, France). We included 45 negative and 44 positive stool samples for Enterocytozoon bieneusi ( n = 34, with various genotypes), Encephalitozoon intestinalis ( n = 4), Encephalitozoon hellem ( n = 4), and Encephalitozoon cuniculi ( n = 2). We also studied a four-year survey of an inter-laboratory quality control program including 9 centers that used this commercial assay. Sensitivity and specificity of the microsporidia generic assay were 86.4% and 93.3%, respectively. Encephalitozoon hellem and Encephalitozoon cuniculi were detected by the microsporidia generic PCR assay but not by the microsporidia typing PCR assay. These results were consistent with the results of the inter-laboratory quality control program. In conclusion, Bio-Evolution Real-time PCR assays are useful tools for intestinal microsporidiosis, but negative results for microsporidia typing assays require supplementary analyses to confirm E. hellem or E. cuniculi infections.Les microsporidioses intestinales sont des infections sous-estimées affectant à la fois les patients immunodéprimés et immunocompétents. Le diagnostic microscopique en laboratoire médical est aujourd’hui supplanté par la PCR en temps réel. Cependant, peu de fabricants incluent les microsporidies dans leurs panels PCR pour le diagnostic des gastro-entérites infectieuses. Ici, nous avons évalué les performances des tests PCR en temps réel microsporidia generic et microsporidia typing (Bio-Evolution, France) sur le thermocycleur PCR en temps réel Rotor-Gene Q (Qiagen, France). Nous avons inclus 45 échantillons de selles négatifs et 44 échantillons positifs pour Enterocytozoon bieneusi ( n = 34, avec divers génotypes), Encephalitozoon intestinalis ( n = 4), Encephalitozoon hellem ( n = 4) et Encephalitozoon cuniculi ( n = 2). Nous avons également analysé les résultats sur 4 ans d’un programme de contrôle qualité inter-laboratoires dont 9 centres ont utilisé ces kits commerciaux. La sensibilité et la spécificité du kit microsporidia generic étaient respectivement de 86,4 % et 93,3 %. Encephalitozoon hellem et E. cuniculi ont été détectés par le kit microsporidia generic mais pas par le kit microsporidia typing. Ces résultats étaient cohérents avec ceux du programme de contrôle de qualité inter-laboratoires. En conclusion, les tests de PCR en temps réel Bio-Evolution sont des outils intéressants pour la microsporidiose intestinale, mais un résultat négatif pour le test de typage microsporidia nécessite une analyse supplémentaire pour confirmer les infections à E. hellem ou E. cuniculi

Deep cutaneous mycoses in kidney transplant recipients: Diagnostic and therapeutic challenges

International audienceDeep cutaneous mycoses (DCMs) are rare infections that extend throughout the dermis and subcutis, often occurring after inoculation with pathogenic fungi. Trends toward a growing incidence have been observed that may be partially related to an increasing population of solid organ transplant patients. The aim of this study is to describe the diagnostics and the outcomes of DCM among kidney transplant recipients so as to optimize their management. We performed a retrospective review of cases of DCM occurring among kidney transplant recipients in our institution over 12 years. Twenty cases were included. Lesions were only located on the limbs and presented mainly as single (10/20, 50%) nodular lesions (15/20, 75%), with a mean size of 3 cm. Direct mycological examination was positive for 17 patients (17/20, 85%) and the cultures were consistently positive. Thirteen different fungal species were observed, including phaehyphomycetes (n = 8), hyalohyphomycetes (n = 3), dermatophytes (n = 1), and mucorale (n = 1). The (1-3) beta-D-glucan antigen (BDG) was also consistently detected in the serum (20/20, 100%). Systematic imaging did not reveal any distant infectious lesions, but locoregional extension was present in 11 patients (11/14, 79%). Nineteen patients received antifungal treatment (19/20, 95%) for a median duration of 3 months, with surgery for 10 (10/20, 50%). There is a great diversity of fungal species responsible for DCMs in kidney transplant recipients. The mycological documentation is necessary to adapt the antifungal treatment according to the sensitivity of the species. Serum BDG positivity is a potentially reliable and useful tool for diagnosis and follow-up