140 research outputs found
Thermogenic Characterization and Antifungal Susceptibility of Candida auris by Microcalorimetry
Candida auris has emerged globally as a multidrug-resistant fungal pathogen. Isolates of C. auris are reported to be misidentified as Candida haemulonii. The aim of the study was to compare the heat production profiles of C. auris strains and other Candida spp. and evaluate their antifungal susceptibility using isothermal microcalorimetry. The minimum heat inhibitory concentrations (MHIC) and the minimum biofilm fungicidal concentration (MBFC) were defined as the lowest antimicrobial concentration leading to the lack of heat flow production after 24 h for planktonic cells and 48 h for biofilm-embedded cells. C. auris exhibited a peculiar heat production profile. Thermogenic parameters of C. auris suggested a slower growth rate compared to Candida lusitaniae and a different distinct heat profile compared to that of C. haemulonii species complex strains, although they all belong to the Metschnikowiaceae clade. Amphotericin B MHIC and MBFC were 0.5 µg/mL and ≥8 µg/mL, respectively. C. auris strains were non-susceptible to fluconazole at tested concentrations (MHIC > 128 µg/mL, MBFC > 256 µg/mL). The heat curve represents a fingerprint of C. auris, which distinguished it from other species. Treatment based on amphotericin B represents a potential therapeutic option for C. auris infection
Development of a bioinformatic tool for the treatment of WGS data for dermatophytes typing and characterization: Focus on squalene epoxidase mutations and terbinafine resistance.
peer reviewedObjectives: The present work aims to use the Whole Genome Sequencing (WGS) as a tool to characterize dermatophytes strains. Data generated by WGS are analyzed by using a bioinformatic tool called “WGS typer” and several markers are highlighted, such as genes implicated in resistance to antifungals or genes linked with high virulence in dermatophytes. The tool will also permit to analyze dermatophytes following their genetic diversity and provide similarity dendrograms. The present work focus on squalene epoxidase (SQLE) gene characterization among T. rubrum and T. indotineae strains by the WGS typer.
Material and methods: 15 strains of T. rubrum (7 resistant to terbinafine and 8 susceptible) and 19 strains of T. indotineae (8 resistant to terbinafine and 11 susceptible) from a multicenter study, previously characterized by Eucast E.Def.11.0 method (Arendrup et al., 2020) were used for SQLE characterization by WGS.WGS has been performed by the GIGA genomics platform using the Illumina technology. The WGS Typer is a commercial bioinformatics tool developed by Hedera-22 (http://www.hedera22.com) and licensed to the Department of Clinical Microbiology of the University of Liège. This tool enables high-throughput typing of pathogen isolates based on raw sequencing data and a collection of relevant markers (single genes, gene variants, gene clusters, MLST). The analysis reports the presence/absence of targeted markers or genotypes from a sequence homology search against the assembled sequencing data according to a set of sequence identity/coverage thresholds.
Results: We evaluated the ability of the tool to detect mutations in the SQLE gene that are responsible for terbinafine resistance in dermatophytes. Seven T. rubrum showed a resistant profile to terbinafine (MIC values >0.25µg/µl) with the microdilution method. Among these, four shared the F397L mutation on SQLE, one was wearing L393F mutation while two other shared the L393S mutation. All these mutations were efficiently highlighted by the WGS typer. Among the eight strains presenting a MIC value under 0.25µg/µl, no mutation was found on SQLE gene. Regarding T. indotineae, 8 strains were previously characterized to be resistant to terbinafine with the microdilution method (MIC values >0,25µg/µl). Among them, the WGS typer detected seven strains with the mutation F397L and one strain with the mutation L393F on the SQLE gene. Among the eleven strains presenting a MIC value under 0.25µg/µl by microdilution, no mutation was found on SQLE. The study was completed with genetic similarity comparisons and dendrogram creation. No clear separation into clusters was observed between resistant/susceptible strains neither in the T. rubrum group nor in the T. indotineae group. T. rubrum and T. indotineae species were well separated into two distinct clusters.
Conclusion: We present here a valuable and innovative tool for the analysis of dermatophytes. The tool permits to easily and accurately detect mutations on the SQLE gene responsible for terbinafine resistance. A dendrogram of similarity based on WGS data can also be generated
The battle against fungi:lessons in antifungal stewardship from COVID 19 times
The COVID-19 pandemic has highlighted the detrimental effect of secondary pathogens in patients with a primary viral insult. In addition to superinfections with bacterial pathogens, invasive fungal infections were increasingly reported. The diagnosis of pulmonary fungal infections has always been challenging; however, it became even more problematic in the setting of COVID-19, particularly regarding the interpretation of radiological findings and mycology test results in patients with these infections. Moreover, prolonged hospitalization in ICU, coupled with underlying host factors. such as preexisting immunosuppression, use of immunomodulatory agents, and pulmonary compromise, caused additional vulnerability to fungal infections in this patient population. In addition, the heavy workload, redeployment of untrained staff, and inconsistent supply of gloves, gowns, and masks during the COVID-19 outbreak made it harder for healthcare workers to strictly adhere to preventive measures for infection control. Taken together, these factors favored patient-to-patient spread of fungal infections, such as those caused by Candida auris, or environment-to-patient transmission, including nosocomial aspergillosis. As fungal infections were associated with increased morbidity and mortality, empirical treatment was overly used and abused in COVID-19-infected patients, potentially contributing to increased resistance in fungal pathogens. The aim of this paper was to focus on essential elements of antifungal stewardship in COVID-19 for three fungal infections, COVID-19-associated candidemia (CAC), -pulmonary aspergillosis (CAPA), and -mucormycosis (CAM).</p
Evaluation multicentrique d'une méthode EUCAST pour tester la sensibilité antifongique des dermatophytes produisant des spores
Background: Terbinafine resistance is increasingly reported in Trichophyton rubrum and Trichophyton interdigitale rendering susceptibility testing important particularly in non-responding cases. We performed a multicentre evaluation of a recently proposed modified EUCAST method implementing medium supplemented with chloramphenicol and cycloheximide (CC) to avoid contamination.
Materials/methods: A blinded panel of wild-type and squalene epoxidase (SQLE) target gene mutant T. rubrum and T. interdigitale strains were distributed to 10 European laboratories. Susceptibility to terbinafine, itraconazole, voriconazole and amorolfine) were performed according to the E.Def 9.3.1 method with and without addition of chloramphenicol and cycloheximide (final concentrations 50 mg/L and 300 mg/L, respectively). Plates were incubated at 25 °C (one laboratory used 30 °C) for 5-7 days until sufficient growth. MICs were determined visually (ignoring trailing growth for itraconazole) and spectrophotometrically with 90% and 50% endpoints yielding a total of 7,829 MICs. A. flavus ATCC 204304 and A. flavus CNM-CM1813 were included as controls.
Results: 100%/96% (voriconazole) and 84%/84% (itraconazole) MIC determinations fell within the QC ranges for the two QC strains, respectively, and 96%/92% terbinafine MICs fell in a 0.25-1 mg/L 3 two-fold-dilution range suggesting a high interlaboratory reproducibility. Across the six methods, the number of terbinafine MEs varied from 2 (2.6%) to 5 (6.6%) for T. rubrum and between 0 and 2 (2.0%) for T. interdigitale (lowest for the CC-method (2.6%-4.4%/ 0-1% for T. rubrum/T. interdigitale). The difference between the modes for the wt and mutant population were ≥7 two-fold-dilutions in all cases (Table). If excluding a I121M/V237I T. rubrum mutant, and two mixed T. interdigitale strains, the number of VMEs were CC visual: T. rubrum: 1/77 (1.3%), CC spec-90%: 3/68 (4.4%) and CC spec-50%: 1/76 (1.3%), and none for T. interdigitale. The activity of voriconazole, itraconazole and amorolfine were quite uniform against T. rubrum and T. interdigitale, but unacceptably wide MIC ranges were found for the visual and spec-90% inhibition methods for itraconazole (data not shown).
Conclusions: Although none of the laboratories perform dermatophyte testing at a regular basis an acceptable interlaboratory agreement and good separation between SQLE wt and mutants were found, suggesting a robust performance of the proposed method
Genotyping of Aspergillus fumigatus in Formalin-Fixed Paraffin-Embedded Tissues and Serum Samples From Patients With Invasive Aspergillosis
Invasive aspergillosis (IA) is a deep tissue infection with a high mortality occurring mostly in immunocompromised patients. To investigate the pathology of patients with IA it may be important to determine the genotype of the invasive isolate of Aspergillus, however available tissues for study are often formalin fixed paraffin embedded (FFPE). Although DNA has been successfully isolated from such tissues for species identification, genotyping of Aspergillus species on such tissues has not yet been performed. In this study we aimed to determine the genotype of Aspergillus fumigatus in FFPE tissue and serum samples from five patients with invasive aspergillosis using nine highly polymorphic short tandem repeat (STRAf) loci. FFPE lung and bronchial biopsies from all patients were successfully typed. By comparing the latter result with non-FFPE materials from non-sterile samples such as sputum, bronchoalveolar lavage and lung abscess, we found identical genotypes within three patients, while the two other patients had a dominant genotype shared among all sample types. Genotyping of serum samples was successful in two serum samples with galactomannan ratios of 4 and 5.6, but failed in serum samples with galactomannan levels <0.5. In addition, testing a subset of these materials with the AsperGenius multiplex qPCR assay, we did not find azole resistance mutations. With this STRAf assay, A. fumigatus from FFPE tissue and serum was successfully genotyped, allowing retrospective examination of A. fumigatus in culture negative patients with IA
Candida auris Identification and Rapid Antifungal Susceptibility Testing Against Echinocandins by MALDI-TOF MS
Candida auris was first reported in an ear swab from Japan in 2009; it then promptly spread over five continents and turned into a global nosocomial problem. The main challenges faced by many researchers are the mis-identification by conventional methods in clinical laboratories and failure in treatment. About 90% of C. auris strains are intrinsically resistant to fluconazole (FLU), and it is developing resistance to multiple classes of available antifungals. Echinocandins are the most potent class of antifungals against C. auris; however, reduced susceptibility to one or many echinocandin drugs has been recently observed. Thus, the main issues addressed in this paper are the fast and accurate identification of C. auris derived from Sabouraud dextrose agar and blood culture bottles as well as the rapid antifungal susceptibility test by MALDI-TOF MS. This study successfully identified all isolates of C. auris (n = 50) by MALDI-TOF MS, with an average log score of ≥ 2. An accuracy of 100% was found on both agar plate and blood culture bottles. MALDI Biotyper antibiotic susceptibility test-rapid assay (MBT ASTRA) was used for rapid antifungal susceptibility testing (AFST). A comparison between MBT ASTRA and the Clinical and Laboratory Standards Institute guidelines (CLSI) detected a sensitivity and specificity of 100% and 98% for anidulafungin, and 100% and 95.5% for micafungin, respectively. A categorical agreement of 98% and 96% was calculated for the two methods. For caspofungin, sensitivity and specificity of 100 and 73% were found, respectively, with a categorical agreement of 82%. MBT ASTRA has the great potential to detect C. auris isolates non-susceptible against echinocandin antifungals within 6 h, which makes it a promising candidate for AFST in clinical laboratories in the future
Azole-Resistance in Aspergillus terreus and Related Species: An Emerging Problem or a Rare Phenomenon?
Raquel Sabino was not included as an author in the published article. It was corrected a posteriori.Erratum in - Corrigendum: Azole-Resistance in Aspergillus terreus and Related Species: An Emerging Problem or a Rare Phenomenon? [Front Microbiol. 2018] Front Microbiol. 2019 Jan 14;9:3245. doi: 10.3389/fmicb.2018.03245. eCollection 2018.Disponível em: https://www.frontiersin.org/articles/10.3389/fmicb.2018.03245/fullFree PMC Article: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5882871/ | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6340063/Objectives: Invasive mold infections associated with Aspergillus species are a significant cause of mortality in immunocompromised patients. The most frequently occurring aetiological pathogens are members of the Aspergillus section Fumigati followed by members of the section Terrei. The frequency of Aspergillus terreus and related (cryptic) species in clinical specimens, as well as the percentage of azole-resistant strains remains to be studied. Methods: A global set (n = 498) of A. terreus and phenotypically related isolates was molecularly identified (beta-tubulin), tested for antifungal susceptibility against posaconazole, voriconazole, and itraconazole, and resistant phenotypes were correlated with point mutations in the cyp51A gene. Results: The majority of isolates was identified as A. terreus (86.8%), followed by A. citrinoterreus (8.4%), A. hortai (2.6%), A. alabamensis (1.6%), A. neoafricanus (0.2%), and A. floccosus (0.2%). One isolate failed to match a known Aspergillus sp., but was found most closely related to A. alabamensis. According to EUCAST clinical breakpoints azole resistance was detected in 5.4% of all tested isolates, 6.2% of A. terreus sensu stricto (s.s.) were posaconazole-resistant. Posaconazole resistance differed geographically and ranged from 0% in the Czech Republic, Greece, and Turkey to 13.7% in Germany. In contrast, azole resistance among cryptic species was rare 2 out of 66 isolates and was observed only in one A. citrinoterreus and one A. alabamensis isolate. The most affected amino acid position of the Cyp51A gene correlating with the posaconazole resistant phenotype was M217, which was found in the variation M217T and M217V. Conclusions:Aspergillus terreus was most prevalent, followed by A. citrinoterreus. Posaconazole was the most potent drug against A. terreus, but 5.4% of A. terreus sensu stricto showed resistance against this azole. In Austria, Germany, and the United Kingdom posaconazole-resistance in all A. terreus isolates was higher than 10%, resistance against voriconazole was rare and absent for itraconazole.This work was supported by ECMM, ISHAM, and EFISG and in
part by an unrestricted research grant through the Investigator
Initiated Studies Programof Astellas, MSD, and Pfizer. This study
was fundet by the Christian Doppler Laboratory for invasive
fungal infections.info:eu-repo/semantics/publishedVersio
Tracing Genetic Exchange and Biogeography of Cryptococcus neoformans var. grubii at the Global Population Level
Cryptococcus neoformans var. grubii is the causative agent of cryptococcal meningitis, a significant source of mortality in immunocompromised individuals, typically human immunodeficiency virus/AIDS patients from developing countries. Despite the worldwide emergence of this ubiquitous infection, little is known about the global molecular epidemiology of this fungal pathogen. Here we sequence the genomes of 188 diverse isolates and characterize the major subdivisions, their relative diversity, and the level of genetic exchange between them. While most isolates of C. neoformans var. grubii belong to one of three major lineages (VNI, VNII, and VNB), some haploid isolates show hybrid ancestry including some that appear to have recently interbred, based on the detection of large blocks of each ancestry across each chromosome. Many isolates display evidence of aneuploidy, which was detected for all chromosomes. In diploid isolates of C. neoformans var. grubii (serotype AA) and of hybrids with C. neoformans var. neoformans (serotype AD) such aneuploidies have resulted in loss of heterozygosity, where a chromosomal region is represented by the genotype of only one parental isolate. Phylogenetic and population genomic analyses of isolates from Brazil reveal that the previously “African” VNB lineage occurs naturally in the South American environment. This suggests migration of the VNB lineage between Africa and South America prior to its diversification, supported by finding ancestral recombination events between isolates from different lineages and regions. The results provide evidence of substantial population structure, with all lineages showing multi-continental distributions; demonstrating the highly dispersive nature of this pathogen
Resistance of Asian Cryptococcus neoformans Serotype A Is Confined to Few Microsatellite Genotypes
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109375.pdf (publisher's version ) (Open Access)BACKGROUND: Cryptococcus neoformans is a pathogenic yeast that causes cryptococcosis, a life threatening disease. The prevalence of cryptococcosis in Asia has been rising after the onset of the AIDS epidemic and estimates indicate more than 120 cases per 1,000 HIV-infected individuals per year. Almost all cryptococcal disease cases in both immunocompromised and immunocompetent patients in Asia are caused by C. neoformans var. grubii. Epidemiological studies on C. neoformans in pan-Asia have not been reported. The present work studies the genetic diversity of the fungus by microsatellite typing and susceptibility analysis of approximately 500 isolates from seven Asian countries. METHODOLOGY/PRINCIPAL FINDINGS: Genetic diversity of Asian isolates of C. neoformans was determined using microsatellite analysis with nine microsatellite markers. The analysis revealed eight microsatellite complexes (MCs) which showed different distributions among geographically defined populations. A correlation between MCs and HIV-status was observed. Microsatellite complex 2 was mainly associated with isolates from HIV-negative patients, whereas MC8 was associated with those from HIV-positive patients. Most isolates were susceptible to amphotericin B, itraconazole, voriconazole, posaconazole, and isavuconazole, but 17 (3.4%) and 10 (2%) were found to be resistant to 5-flucytosine and fluconazole, respectively. Importantly, five Indonesian isolates (approximately 12.5% from all Indonesian isolates investigated and 1% from the total studied isolates) were resistant to both antifungals. The majority of 5-flucytosine resistant isolates belonged to MC17. CONCLUSIONS: The findings showed a different distribution of genotypes of C. neoformans var. grubii isolates from various countries in Asia, as well as a correlation of the microsatellite genotypes with the original source of the strains and resistance to 5-flucytosine
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