Rachid Zouhair

Genotyping Study of Scedosporium apiospermum Isolates from Patients with Cystic Fibrosis

ABSTRACT Usually a saprophyte, Scedosporium apiospermum often colonizes the respiratory tracts of patients with cystic fibrosis (CF). In order to improve our understanding of the molecular epidemiology of the airway colonization, 129 sequential and multiple isolates collected from January 1998 to March 1999 from nine CF patients monitored in three hospitals in France were typed by random amplification of polymorphic DNA with primers GC70, UBC-701, and UBC-703. Among these primers, UBC-703 was the most discriminating, allowing the differentiation of 14 genotypes. Combining the results obtained with this three-primer set resulted in the differentiation of 16 genotypes. No common genotype was found among the different patients, and no clustering according to geographic origin of the isolates was seen. In addition, five of the patients were colonized by a single genotype. The others usually exhibited a predominant genotype accompanied by one or two others, which were found occasionally and were genetically close to the predominant genotype. Thus, our study demonstrates the persistence of the fungus despite antifungal treatments and therefore reinforces the need for the development of new antifungals that are more efficient against this species.

In-vivo selection of an azole-resistant petite mutant of Candida glabrata

Two isolates of Candida glabrata from the same stool sample from a bone marrow transplant recipient treated with fluconazole, and designated 1084-L for large colonies on yeast extract-peptone-dextrose-agar and 1084-S for small colonies, were analysed. In-vitro susceptibility tests with a commercially available disk diffusion procedure showed that isolate 1084-L had a susceptibility pattern typical of wild-type strains of C. glabrata with sensitivity to polyenes and the presence of resistant colonies randomly distributed within the inhibition zones for all azole compounds except tioconazole. In contrast, isolate 1084-S, which was found by pulsed-field gel electrophoresis and random amplification of polymorphic DNA to be genetically closely related to isolate 1084-L, exhibited cross-resistance to the azole compounds except tioconazole. Determination of MICs by the E-test method confirmed these results, showing that isolate 1084-S had greater sensitivity to amphotericin B and complete resistance to ketoconazole and fluconazole. Growth on agar plates containing glucose or glycerol as the sole carbon source suggested that the resistant isolate had a respiratory deficiency, which was further demonstrated by flow cytometric analysis of the fluorescence of rhodamine 123-stained blastoconidia. Restriction endonuclease analysis of mitochondrial DNA (mtDNA) established the mitochondrial origin of the respiratory deficiency. However, PCR amplification of the mtDNA with primers ML1 and ML6, as well as transmission electron microscopy, suggested a partial deletion of the mtDNA analogous to that described for rho- petite mutants of Saccharomyces cerevisiae. Together, these results provided evidence that the selection of azole-resistant petite mutants of C. glabrata may occur in vivo after fluconazole administration, which might explain, therefore, clinical failure of antifungal therapy.

Molecular epidemiology of airway colonisation by Aspergillus fumigatus in cystic fibrosis patients.

A total of 109 sequential and multiple Aspergillus fumigatus isolates corresponding to 41 samples from seven cystic fibrosis (CF) patients was typed by random amplification of polymorphic DNA (RAPD) with the primer NS3 from the fungal ribosomal gene 18S subunit, and by sequence-specific DNA primer (SSDP) analysis. RAPD typing of the isolates revealed 10 different genotypes, whereas nine genotypes were identified by SSDP. Combination of the two typing methods permitted the differentiation of 25 overall genotypes. The colonisation typing patterns differed greatly between patients colonised for <1 year by A. fumigatus and long-term colonised patients. Two of three recently colonised patients presented a large number of types even in the same sample, unlike the chronically colonised patients, who harboured a limited number of genotypes. In the latter, the occurrence of a dominant genotype, usually the overall genotype 2, tended to reflect to the duration of colonisation. Moreover, anti-catalase antibodies to A. fumigatus appeared in most cases to be in response to genotype 2. These findings suggest that some strains of A. fumigatus may be selected during prolonged colonisation of the airways in CF patients.

Distribution of the different species of the Pseudallescheria boydii/Scedosporium apiospermum complex in French patients with cystic fibrosis.

As various new sibling species within the Pseudallescheria boydii/Scedosporium apiospermum complex have been described recently with differences in their susceptibility to antifungals, this study was conducted in order to determine their respective frequency in cystic fibrosis. Results indicated that P. boydii largely predominated (62%), followed by S. apiospermum (24%), Scedosporium aurantiacum (10%) and Pseudallescheria minutispora (4%). Scedosporium dehoogii was not recovered in this study. The multiple correspondence factor analysis highlighted geographical discrepancies within species distribution: P. boydii was rarely encountered in Northern France, while S. apiospermum was less represented in the west of the country. Additionally, we demonstrated that all species encountered in the cystic fibrosis context were capable to chronically colonize the respiratory tract of patients. Molecular typing of a large set of environmental and clinical isolates should be conducted to delineate the epidemiology of each sibling species in the complex.

Clinical and microbiological efficacy of micafungin on Geosmithia argillacea infection in a cystic fibrosis patient

Cystic fibrosis (CF) patients are at high risk of colonization of the airways by a number of fungi, including the emerging opportunistic fungus Geosmithia argillacea. We report the eradication of respiratory G. argillacea associated with clinical resolution of severe symptoms by high-dose and prolonged micafungin therapy in a young CF patient.

Semi-automated repetitive sequence-based PCR amplification for species of the Scedosporium apiospermum complex

PURPOSE The Scedosporium apiospermum species complex usually ranks second among the filamentous fungi colonizing the airways of patients with cystic fibrosis (CF), but little is known about the molecular epidemiology of the airway colonization. METHODS Polymerase chain reaction (PCR) amplification of repetitive sequences (rep-PCR) was applied to the retrospective analysis of a panel of isolates already studied by random amplification of polymorphic DNA (RAPD) and comprising 63 isolates recovered from sputa from 9 CF patients. Results were compared to those obtained previously by RAPD, and herein by beta-tubulin (TUB) gene sequencing and Multilocus Sequence Typing (MLST). RESULTS Within the panel of isolates studied,S. apiospermum sensu stricto and Scedosporium boydii, as expected, were the predominant species with 21 and 36 isolates, respectively. Four isolates from one patient were identified as Scedosporium aurantiacum, whereas two isolates belonged to the Pseudallescheria ellipsoidea subgroup of S. boydii rep-PCR analysis of these isolates clearly differentiated the three species and P. ellipsoidea isolates, whatever the rep-PCR kit used, and also permitted strain differentiation. When using the mold primer kit, results from rep-PCR were in close agreement with those obtained by MLST. For both S. apiospermum and S. boydii, 8 genotypes were differentiated by rep-PCR and MLST compared to 10 by RAPD. All S. aurantiacum isolates shared the same RAPD genotype and exhibited the same rep-PCR profile and sequence type. CONCLUSIONS These results illustrate the efficacy of rep-PCR for both species identification within the S. apiospermum complex and genotyping for the two major species of this complex.Abstract presentation: Part of this work was presented during the 18th Congress of the International Society for Human and Animal Mycology, Berlin (Germany), June 2012.S. Giraud, C. Godon, A. Rougeron, J.P. Bouchara and L. Favennec are members of the ECMM/ISHAM working group on Fungal respiratory infections in Cystic Fibrosis(Fri-CF).

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

ABSTRACT 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.

Prevalence of the sibling species of the Scedosporium apiospermum complex in cystic fibrosis context

O1.1 Risk factors for invasive fungal infection among patients with hematologic malignancies M. Hoenigl, H. J. Salzer, A. Strohmaier, T. Valentin, I. Zollner-Schwetz, A. Rohn, K. Seeber, A. J. Grisold, W. Linkesch and R. Krause Section of Infectious Diseases, Medical University of Graz, Austria, Division of Hematology, Medical University of Graz, Austria, AGES PharmMed and Federal Office for Safety in Health.Care, Austrian Agency for H, Austria and Medical university of Graz, Austria