Epidemiology and reporting of candidaemia in Belgium: a multi-centre study.

The primary aim of this study was to collect national epidemiological data on candidaemia and to determine the reporting time of species identification and antifungal susceptibility in clinical practice. During a 1-year period (March 2013 until February 2014), every first Candida isolate from each episode of candidaemia was included prospectively from 30 Belgian hospitals. Identification and susceptibility testing were performed according to local procedures and isolates were sent to the National Reference Center for Mycosis. Species identification was checked by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and internal transcribed spacer (ITS) sequencing in case no reliable identification was obtained by MALDI-TOF MS. Antifungal susceptibility testing was performed according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) methodology. A total of 355 isolates were retrieved from 338 patients. The mean incidence rate of candidaemia was 0.44 (range: 0.07 to 1.43) per 1000 admissions or 0.65 (range: 0.11 to 2.00) per 10,000 patient days. Candida albicans was most frequently found (50.4 %), followed by C. glabrata (27.3 %) and C. parapsilosis sensu lato (9.8 %). The overall resistance to fluconazole was 7.6 %, ranging from 3.9 % in C. albicans to 20.0 % in C. tropicalis. Only one C. glabrata isolate was resistant to the echinocandins. Four days after blood culture positivity, 99.7 % of the identifications and 90.3 % of the antifungal profiles were reported to the treating clinician. Candidaemia incidence rates differed up to 20-fold among Belgian hospitals; no clear factors explaining this difference were identified. The overall antifungal resistance rates were low but high azole resistance rates were recorded in C. tropicalis.

Mechanisms of skin adherence and invasion by dermatophytes.

Dermatophytes are keratinophilic fungi that can be pathogenic for humans and animals by infecting the stratum corneum, nails, claws or hair. The first infection step consists of adherence of arthroconidia to the stratum corneum. The mechanisms and the kinetics of adherence have been investigated using different in vitro and ex vivo experimental models, most notably showing the role of a secreted serine protease from Microsporum canis in fungal adherence to feline corneocytes. After germination of the arthroconidia, dermatophytes invade keratinised structures that have to be digested into short peptides and amino acids to be assimilated. Although many proteases, including keratinolytic ones, have been characterised, the understanding of dermatophyte invasion mechanisms remains speculative. To date, research on mechanisms of dermatophyte infection focused mainly on both secreted endoproteases and exoproteases, but their precise role in both fungal adherence and skin invasion should be further explored.

Comparison of multiple typing methods for Aspergillus fumigatus.

As part of studies on the spread of infections, risk factors and prevention, several typing methods were developed to investigate the epidemiology of Aspergillus fumigatus. In the present study, 52 clinical isolates of A. fumigatus from 12 airway specimens from patients with invasive aspergillosis (hospitalized in three different centres) were characterized by short tandem repeat (STR) typing and multilocus sequence typing (MLST). These isolates were previously typed by random amplified polymorphic DNA (RAPD), sequence-specific DNA polymorphism (SSDP), microsatellite polymorphism (MSP) and multilocus enzyme electrophoresis (MLEE). STR typing identified 30 genotypes and, for most patients, all isolates were grouped in one cluster of the unweighted pair group method with arithmetic mean dendrogram. Using MLST, 16 genotypes were identified among 50 isolates, while two isolates appeared untypeable. RAPD, MSP, SSDP and MLEE allowed identification of eight, 14, nine and eight genotypes, respectively. Combining the results of these methods led to the delineation of 25 genotypes and a similar clustering pattern as with STR typing. In general, STR typing led to similar results to the previous combination of RAPD, SSDP, MSP and MLEE, but had a higher resolution, whereas MLST was less discriminatory and resulted in a totally different clustering pattern. Therefore, this study suggests the use of STR typing for research concerning the local epidemiology of A. fumigatus, which requires a high discriminatory power.

Use of mass spectrometry to identify clinical Fusarium isolates.

Fusarium spp. have recently emerged as significant human pathogens. Identification of these species is important, both for epidemiological purposes and for patient management, but conventional identification based on morphological traits is hindered by major phenotypic polymorphism. In this study, 62 strains, or isolates, belonging to nine Fusarium species were subjected to both molecular identification TEF1 gene sequencing and matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) analysis. Following stringent standardization, the proteomic-based method appeared to be both reproducible and robust. Mass spectral analysis by comparison with a database, built in this study, of the most frequently isolated species, including Fusarium solani, Fusarium oxysporum, Fusarium verticilloides, Fusarium proliferatum and Fusarium dimerum, correctly identified 57 strains. As expected, the four species (i.e. Fusarium chlamydosporum, Fusarium equiseti, Fusarium polyphialidicum, Fusarium sacchari) not represented in the database were not identified. Results from mass spectrometry and molecular identification agreed in five of the six cases in which results from morphological and molecular identification were not in agreement. MALDI-TOF yielded results within 1 h, making it a valuable tool for identifying clinical Fusarium isolates at the species level. Uncommon species must now be added to the database. MALDI-TOF may also prove useful for identifying other clinically important moulds.

High-resolution genotyping of Aspergillus fumigatus isolates recovered from chronically colonised patients with cystic fibrosis.

A series of 256 Aspergillus fumigatus isolates, recovered from eight patients with cystic fibrosis (CF), were genotyped using microsatellite-based typing. Only a limited number of genotypes were shared between patients and co-colonisation with multiple strains was indicated for all patients. Additionally, some genotypes were isolated recurrently, indicating that they are capable of prolonged colonisation.

Molecular identification of Cryptococcus neoformans serotypes.

Cryptococcus neoformans is a fungal pathogen that causes life-threatening infections primarily in immunocompromised hosts. Based on the genetic characteristics and serologic properties of capsular polysaccharides, three varieties and five serotypes have been defined: C. neoformans var. neoformans (serotype D), C. neoformans var. grubii (serotype A), hybrid serotype AD, and C. neoformans var. gattii (serotypes B and C). Epidemiologic features, such as geographic distribution and ecologic niche, and clinical characteristics have been shown to be associated with serotypes. At the present time, serotyping is based on agglutination tests with either commercial or "homemade" antisera or on immunofluorescence assays using a monoclonal antibody directed against the capsule polysaccharide. In this paper, we describe two molecular methods (PCR-restriction enzyme analysis and length polymorphism analysis) for C. neoformans serotype identification. Both are based on the sequence characteristics of a fragment of the CAP59 gene required for capsule biosynthesis. Testing of 72 C. neoformans strains including representatives of the five serotypes demonstrated the reliability of these methods.

Environmental investigations and molecular typing of Aspergillus flavus during an outbreak of postoperative infections.

After an outbreak of sternal surgical-site infections (SSSI) with Aspergillus flavus following cardiac surgery, a mycological survey of air and surfaces (41 and 149 samples, respectively) was performed throughout the surgical ward (SW) and in other areas of the hospital. Results showed massive contamination by A. flavus: more than 100 cfu per contact plate were frequently observed in some areas of the SW. The distribution of the A. flavus spores in the building, and especially in the SW, enabled the location of a possible source within the non-medical part of the SW, but the true source could not be identified. Four other surveys were made to follow up the decontamination process; the contamination level did not fall rapidly, needing repetitive cleaning operations. Strains from patients and from the hospital environment selected all over the SW were typed by random amplification of polymorphic DNA (RAPD), using two different primers (ERIC-1, BG-2). All these strains showed the same genotype, proving the clonal single-source of the environmental contamination and the intra-operative acquisition of A. flavus in the SSSI outbreak.

Hospital-acquired Aspergillus fumigatus infection: can molecular typing methods identify an environmental source?

Aspergillus fumigatus infection in hospitalized immunocompromised patients often raises suspicion regarding the potential for hospital acquisition. Hospital staff have an important responsibility in implementing preventive measures, especially since the advent of current legislation concerning hospital-acquired infections. There have been high expectations that molecular typing methods might determine the source of Aspergillus fumigatus, a ubiquitous mould. The aim of the present epidemiological study, was therefore, to identify the origin(s) of Aspergillus infection in six well-documented patients. All the clinical strains (N=33), and those from hospital (N=14) and home environments (N=34) were isolated according to a standardized protocol and typed by sequence-specific DNA primer analysis. The results confirmed the huge biodiversity of the A. fumigatus population, and consequently the difficulty in ascertaining a hospital source of the infection, as opposed to infections due to other Aspergillus species less frequently encountered.

Prevalence of Candida dubliniensis in the BCCM/IHEM Biomedical Fungi/Yeasts culture collection (isolates before 1990).

The BCCM/IHEM Biomedical Fungi/Yeasts collection hosts 1200 Candida albicans strains of the Vanbreuseghem mycotheque isolated between 1951 and 1997. From this collection, 469 freeze-dried C. albicans strains, producing chlamydospores, germ tubes and forming green colonies on CHROMagar, all isolated before 1990, were screened to identify the Candida dubliniensis isolates. Screening was performed in different steps using the growth at 45 degrees C, the assimilation of xylose, the intracellular beta-glucosidase activity test and C. dubliniensis-specific polymerase chain reaction (PCR) with primers from ACT1 intron sequence. Five isolates (1%) were identified as C. dubliniensis: one isolate was not documented, the others were of oropharyngeal origin of which two (1987 and 1990) were from proven human immunodeficiency virus patients.

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.

Genetic polymorphism of Aspergillus fumigatus in clinical samples from patients with invasive aspergillosis: investigation using multiple typing methods.

The genotypes of 52 strains of Aspergillus fumigatus isolated 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.

Molecular typing of Aspergillus terreus isolates by random amplification of polymorphic DNA.

Forty three isolates of Aspergillus terreus of environmental or clinical origin were typed by random amplification of polymorphic DNA (RAPD) with two different primers NS3 and NS7 from the fungal ribosomal 18S subunit gene. For the 31 epidemiologically unrelated isolates tested, the primers NS3 and NS7 gave rise to 23 and 24 different genotypes, respectively, and combining the results obtained with the two primers allowed the differentiation of all these isolates. No clustering was found in relation to pathogenicity, clinical signs, or geographic origin of the isolates. Five groups of related isolates of A. terreus were also typed. Analysis of sequential isolates from patients with cystic fibrosis or with invasive aspergillosis showed the clonality of the colonization or infection by A. terreus. Likewise, this straightforward typing method demonstrated the clonal origin of a massive contamination of the environment in a haematology unit. Therefore this RAPD typing method may constitute a valuable tool for the epidemiological follow-up of airway colonization in patients with cystic fibrosis or investigations of links between nosocomial outbreaks of invasive aspergillosis and environmental contamination.

Nosocomial pseudo-outbreak of Fusarium verticillioides associated with sterile plastic containers.

Twenty-six patients were implicated in a nosocomial pseudo-outbreak of Fusarium verticillioides. Examination of clinical records and handling procedures revealed a fungal contamination of supposedly sterile containers used for biological materials. An accurate system of monitoring permitted us to determine the origin of the infection and the means of its spread.

Identification of Fusarium species involved in human infections by 28S rRNA gene sequencing.

Fusarium spp. have emerged as major opportunistic fungal agents. Since new antifungal agents exhibit variable activity against Fusarium isolates depending on the species, rapid identification at the species level is required. Conventional culture methods are difficult, fastidious, and sometimes inconclusive. In this work, we sequenced a 440-bp fragment encoding the 28S rRNA from 33 Fusarium isolates belonging to six Fusarium species associated with human infections. The data were then analyzed by the neighbor-joining method. By using distance matrix analysis and constructing the phylogram, we could easily distinguish the different species for all but one isolate. The method also allowed differentiation between the closely related genera Acremonium and Cylindrocarpon. In contrast to the case with conventional methods, the results could be obtained within 48 h from a 3-day culture and are independent of mycologist experience, making this method rapid and reliable for identification of Fusarium species isolated from patients.

Combination of three typing methods for the molecular epidemiology of Aspergillus fumigatus infections. European Research Group on Biotype and Genotype of Aspergillus.

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 patient's 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.

Molecular typing of Aspergillus fumigatus strains by sequence-specific DNA primer (SSDP) analysis.

A PCR typing method has been developed and tested to investigate the polymorphism of clinical strains of Aspergillus fumigatus. Firstly, the DNA fragments from random amplified polymorphic DNA (RAPD) patterns of nine epidemiologically and geographically non-related monosporal strains of A. fumigatus were cloned and sequenced. The pairs of five sequence-specific DNA primers (SSDP), characteristic of the 5' and 3' extremities of the RAPD products, were then used in high stringency PCR to type 43 clinical strains of A. fumigatus from 13 patients, according to the presence or absence of a single amplified band. This original approach, which uses the advantages of PCR, has made it possible to overcome the difficulties resulting from the low stringency amplification. The SSDP analysis of 51 A. fumigatus strains (9 unrelated monosporal strains and 43 clinical strains from 13 patients) can be classed into 22 different types with a high reproducibility and a high level of discrimination (D = 0.96). The results suggest that seven lung transplant patients with necrotizing aspergillosis, bronchitis aspergillosis and bronchial colonization were infected by multiple strain genotypes, whereas three patients with invasive aspergillosis seem to have been infected by a single strain.

Multicentric epidemiological study of Aspergillus fumigatus isolates by multilocus enzyme electrophoresis.

The genotypes of 63 isolates of Aspergillus fumigatus obtained from three hospitals in different geographical areas and of eight culture collection strains were determined by multilocus enzyme electrophoresis. Twelve of the 17 enzymatic loci studied were polymorphic, giving rise to 48 different electrophoretic types. The existence of fixed multilocus genotypes, significant heterozygote deficits and excesses at the different loci, and linkage disequilibria within subpopulations strongly suggests a clonal reproduction mode for A. fumigatus. Numerical analysis of the comparison and disposition of the different electrophoretic types demonstrates a significant genetic differentiation between the three sampling sites. However, no correlation could be found between geographical distances and genetic differentiation. On account of the multiple discriminatory markers, multilocus enzyme electrophoresis typing seems to be a very powerful tool for epidemiological and reproductive mode studies of A. fumigatus.