Sébastien Bertout

Genetic Polymorphism of Aspergillus fumigatus in Clinical Samples from Patients with Invasive Aspergillosis: Investigation Using Multiple Typing Methods

ABSTRACT The genotypes of 52 strains of Aspergillus fumigatusisolated from 12 patients with invasive aspergillosis were investigated using three typing methods (random amplified polymorphic DNA, sequence-specific DNA polymorphism, and microsatellite polymorphism) combined with multilocus enzyme electrophoresis. Isolates were from patients hospitalized in three different geographic areas (Lyon, France; Grenoble, France; and Milan, Italy). In each case, the genetic polymorphism of several colonies (two to five) within the first respiratory clinical sample was studied. For the 52 isolates tested, random amplified polymorphic DNA identified 8 different genotypes, sequence-specific DNA polymorphism identified 9 different types, and microsatellite polymorphism identified 14 types. A combination of these results with multilocus enzyme electrophoresis study identified 25 different types within the sample studied. We identified 3 patients (of the 12 studied) who carried a single genotype; 6 patients were infected by two genotypes, 1 patient had four genotypes, while the last patient had five. A combination of typing methods provided better discrimination than the use of a single method. Typing methods revealed a population structure within each geographical site, suggesting that the epidemiology of A. fumigatus should be considered separately for each of these geographic areas. This study demonstrates the usefulness of combining several typing methods in reaching an understanding of the epidemiology of A. fumigatus and clarifies whether it is sufficient to type one isolate from each specimen to determine the strain involved in invasive aspergillosis.

Environmental distribution of Cryptococcus neoformans and C. gattii around the Mediterranean basin

In order to elucidate the distribution of Cryptococcus neoformans and C. gattii in the Mediterranean basin, an extensive environmental survey was carried out during 2012-2015. A total of 302 sites located in 12 countries were sampled, 6436 samples from 3765 trees were collected and 5% of trees were found to be colonized by cryptococcal yeasts. Cryptococcus neoformans was isolated from 177 trees and C. gattii from 13. Cryptococcus neoformans colonized 27% of Ceratonia, 10% of Olea, Platanus and Prunus trees and a lower percentage of other tree genera. The 13 C. gattii isolates were collected from five Eucalyptus, four Ceratonia, two Pinus and two Olea trees. Cryptococcus neoformans was distributed all around the Mediterranean basin, whereas C. gattii was isolated in Greece, Southern Italy and Spain, in agreement with previous findings from both clinical and environmental sources. Among C. neoformans isolates, VNI was the prevalent molecular type but VNII, VNIV and VNIII hybrid strains were also isolated. With the exception of a single VGIV isolate, all C. gattii isolates were VGI. The results confirmed the presence of both Cryptococcus species in the Mediterranean environment, and showed that both carob and olive trees represent an important niche for these yeasts.

Typing of Saccharomyces cerevisiae Clinical Strains by Using Microsatellite Sequence Polymorphism

ABSTRACT It seems that S. cerevisiae, which was thought for about 30 years to be a nonpathogenic yeast, should now be considered an opportunistic pathogen. In this study, we estimated the discrimination ability of the microsatellite sequence amplification technique within a sample of clinical and reference S. cerevisiae strains and S. boulardii reference strains.

Taxonomy and antifungal susceptibility of clinically important Rasamsonia species

ABSTRACT In recent years, Geosmithia argillacea has been increasingly reported in humans and animals and can be considered an emerging pathogen. The taxonomy of Geosmithia was recently studied, and Geosmithia argillacea and related species were transferred to the new genus Rasamsonia. The diversity among a set of Rasamsonia argillacea strains, including 28 clinical strains, was studied, and antifungal susceptibility profiles were generated. Data obtained from morphological studies and from phylogenetic analyses of internal transcribed spacer (ITS) and partial β-tubulin and calmodulin sequences revealed the presence of four species in the Rasamsonia argillacea complex, two of which are newly described here: R. piperina sp. nov. and R. aegroticola sp. nov. In contrast to other related genera, all Rasamsonia species can be identified with ITS sequences. A retrospective identification was performed on recently reported clinical isolates from animal or human patients. Susceptibility tests showed that the antifungal susceptibility profiles of the four members of the R. argillacea complex are similar, and caspofungin showed significant activity in vitro, followed by amphotericin B and posaconazole. Voriconazole was the least active of the antifungals tested. The phenotypically similar species R. brevistipitata and R. cylindrospora had different antifungal susceptibility profiles, and this indicates that correct species identification is important to help guide appropriate antifungal therapy.

Importance of Resolving Fungal Nomenclature: the Case of Multiple Pathogenic Species in the Cryptococcus Genus

Cryptococcosis is a major fungal disease caused by members of the Cryptococcus gattii and Cryptococcus neoformans species complexes. After more than 15 years of molecular genetic and phenotypic studies and much debate, a proposal for a taxonomic revision was made. ABSTRACT Cryptococcosis is a major fungal disease caused by members of the Cryptococcus gattii and Cryptococcus neoformans species complexes. After more than 15 years of molecular genetic and phenotypic studies and much debate, a proposal for a taxonomic revision was made. The two varieties within C. neoformans were raised to species level, and the same was done for five genotypes within C. gattii. In a recent perspective (K. J. Kwon-Chung et al., mSphere 2:e00357-16, 2017, https://doi.org/10.1128/mSphere.00357-16 ), it was argued that this taxonomic proposal was premature and without consensus in the community. Although the authors of the perspective recognized the existence of genetic diversity, they preferred the use of the informal nomenclature “C. neoformans species complex” and “C. gattii species complex.” Here we highlight the advantage of recognizing these seven species, as ignoring these species will impede deciphering further biologically and clinically relevant differences between them, which may in turn delay future clinical advances.

A longitudinal study of lung transplant recipients infected with Aspergillus: genetic polymorphism of A fumigatus

BACKGROUND Aspergillus infection is a well-known complication of lung transplantation and remains associated with high mortality rates. Molecular typing methods are required to elucidate the complex epidemiology of Aspergillus disease in lung transplant recipients. METHODS Eight lung transplant recipients from one hospital were followed for A fumigatus colonization or infection. Forty-four sequential isolates from these patients were selected and typed by three molecular methods (random amplified polymorphic DNA, sequence-specific DNA primer and multi-locus enzyme electrophoresis). RESULTS Sixteen different types were identified of which 14 were specific to 1 patient. A factorial correspondence analysis showed that variability between sequential isolates from a single patient was as high as between isolates from the other patients. Lung transplant recipients presented many different genotypes, reflecting the environmental diversity of A fumigatus. Nevertheless, throughout their follow-up, 2 of the 8 lung transplant recipients harbored a common genotype that was not replaced by others. CONCLUSIONS These results confirm the important genetic polymorphism of the A fumigatus population. The observed genotypes were not related to the type of Aspergillus disease or anti-fungal treatment used nor to the outcome of the patient. These data confirm that all A fumigatus molecular types present the same pathogenic risk.

Investigation of minor species Candida africana, Candida stellatoidea and Candida dubliniensis in the Candida albicans complex among Yaoundé (Cameroon) HIV‐infected patients

Minor species of the Candida albicans complex may cause overestimation of the epidemiology of C. albicans, and misidentifications could mask their implication in human pathology. Authors determined the occurrence of minor species of the C. albicans complex (C. africana, C. dubliniensis and C. stellatoidea) among Yaoundé HIV‐infected patients, Cameroon. Stool, vaginal discharge, urine and oropharyngeal samples were analysed by mycological diagnosis. Isolates were identified by conventional methods and mass spectrometry (MS; carried out by the matrix‐assisted laser desorption–ionisation time‐of‐flight MS protocol). Candida albicans isolates were thereafter submitted to the PCR amplification of the Hwp1 gene. The susceptibility of isolates to antifungal drugs was tested using the Clinical and Laboratory Standards Institute M27‐A3 protocol. From 115 C. albicans obtained isolates, neither C. dubliniensis nor C. stellatoidea was observed; two strains of C. africana (422PV and 448PV) were identified by PCR electrophoretic profiles at 700 bp. These two C. africana strains were vaginal isolates. The isolate 448PV was resistant to ketoconazole at the minimal inhibitory concentration of 2 μg ml−1, and showed reduced susceptibility to amphotericin B at 1 μg ml−1. This first report on C. africana occurrence in Cameroon brings clues for the understanding of the global epidemiology of this yeast as well as that of minor species of the C. albicans complex.

Multilocus enzyme electrophoresis analysis of Aspergillus fumigatus strains isolated from the first clinical sample from patients with invasive aspergillosis

The genotypes of 50 isolates of Aspergillus fumigatus from 11 patients with invasive aspergillosis, obtained from three hospitals in different geographical areas, were determined by multilocus enzyme electrophoresis (MLEE). The study analysed the genetic polymorphism of multiple isolates from the first sample. Seven of the 14 enzymic loci studied were polymorphic, giving rise to eight different electrophoretic types. For nine of 11 patients studied, no polymorphism was observed in isolates within the first clinical sample. Analysis of genetic distance between electrophoretic types demonstrated a genetic heterogeneity within each geographical site. Moreover, some genotypes were preferentially found in a given area and this revealed a population structure within these geographical sites. Therefore, the epidemiology of A. fumigatus should be considered separately for each of these areas. The multiple discriminatory markers of MLEE seem to provide a powerful tool for increasing the understanding of the biology of this fungus.

A Candida albicans strain with high MIC for caspofungin and no FKS1 mutations exhibits a high chitin content and mutations in two chitinase genes

We studied the cell wall of a Candida albicans laboratory mutant exhibiting a high minimum inhibitory concentration (MIC; 8 μg ml(-1)) for caspofungin without bearing FKS1 mutations. This strain showed a reduced level of ß 1,3 D glucan (0.43×) and a higher chitin content (2.3×) than a control strain even when grown without caspofungin. No significant over- or under-expression of chitin synthase or chitinase genes was observed. However, point mutations were detected in the chitinase 2 and 3 genes. These mutations, which may affect the enzymatic activity of the encoded protein products involved in the degradation of the chitin, could have led to an increased concentration of that component, allowing the strain to compensate for its low ß 1,3 D glucan content and the effect of caspofungin.

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.