M. Mallié

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.

Atypical strains of Candida albicans recovered from AIDS patients

By using multilocus enzyme electrophoresis (MLEE) we have analyzed the genetic diversity encountered among chlamydospore-positive Candida albicans strains. While the type II strains of the former C. stellatoidea were genetically indistinguishable from those of C. albicans, type I strains constituted a distinct subgroup compared with C. albicans strains. Nevertheless, all these strains remained genetically very closely related compared with other species of Candida (e.g. C. tropicalis, C. krusei and C. glabrata). These results corroborate the synonymy between C. stellatoidea and C. albicans. Chlamydospore-positive C. albicans strains with atypical sugar assimilation patterns displayed a great genetic divergence from the cluster constituted by C. albicans and the strains of the former C. stellatoidea. However, these atypical strains were more closely related to C. albicans than they were to C. tropicalis, C. krusei or C. glabrata. These strains represent a genetically entity distinct from the typical C. albicans strains used in this study. The data also support the view that the atypical strains described here belong to the same genetic group as atypical C. albicans strains previously described by others.

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.

Genetic Structure of Candida glabrata Populations in AIDS and Non-AIDS Patients

ABSTRACT The genotypes of 63 strains (11 reference strains and 52 strains from hospitalized patients) of the haploid yeast Candida glabrata were determined from 33 putative gene enzymatic loci. This enabled the characterization of 26 different multilocus genotypes. Genetic differentiation was found between distant hospitals (located in Montpellier and Paris, France) but not for other parameters (anatomic origins or human immunodeficiency virus-positive [HIV+] and HIV− patients). Strong nonrandom association between loci could be seen. Such statistical linkages were confirmed upon comparing the patterns of 14 RAPD [random(ly) amplified polymorphic DNA] primers from 20 of these strains to results obtained from multilocus enzyme electrophoresis analysis. This finding suggests a mainly clonal mode of reproduction of C. glabrata. The consequences of the clonality displayed by C. glabrata populations on the epidemiology of this yeast are also discussed.

Combination of three typing methods for the molecular epidemiology of Aspergillus fumigatus infections

This study investigated the source of infection and strain relatedness of Aspergillus fumigatus isolates from bronchial colonisation and invasive aspergillosis (IA) in four transplant patients. Environmental isolates from the patients home and from the hospital and infecting isolates were obtained for patient A who developed IA. Clinic environmental and colonising isolates were obtained for patient B. Sequential isolates were obtained from various organs from patient C who developed IA and also from patient D who had a bronchitic aspergillosis that developed into IA. Ninety-one A. fumigatus isolates were analysed by three typing methods: multi-locus enzyme electrophoresis (MLEE), random amplified polymorphic DNA (RAPD) and sequence-specific DNA primers (SSDP). The three combined typing methods demonstrated a greater differentiation of isolates than the typing methods used separately or in pairs. This demonstrated the genotypic variability of A. fumigatus and facilitated better epidemiological analysis. Large polymorphisms were demonstrated for each patient isolate between and colonies within various samples. The relatedness of the isolates suggested nosocomially acquired aspergillosis for patient B, but the source of infection for patient A remained unclear. The results suggested at least three multiple infections among the four patients. This study enabled the identification of the source of infection and strain relatedness, which in turn facilitates the development of preventive measures for patient management in the future.

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.

Multicentric genetic study of Candida albicans isolates from non-neutropenic patients using MLEE typing: population structure and mode of reproduction.

A mycological survey was conducted on non‐neutropenic patients in three distinct intensive care units in two hospitals in Marseille (France) from November 1993 to November 1995. Candida albicans positive cultures from 62 patients were included in this study. Every first isolate of each patient was typed by multilocus enzyme electrophoresis (MLEE). The enzyme profiles obtained from 15 polymorphic loci were then compared. This analysis demonstrated a strong population differentiation of C. albicans infective strains within and between the different care units and confirmed the probable preponderant clonal mode of reproduction of this yeast.