Multiple genotyping studies have been carried out in order to clarify the epidemiology of fungal infections, more specifically to determine the sources, transmission routes, and colonization patterns of fungal isolates. In this review, the results obtained in genotyping investigations of Aspergillus isolates are summarized and discussed. Furthermore, we examine the epidemiologic studies of Candida albicans, Exophiala dermatitidis and Scedosporium apiospermum infections in patients with cystic fibrosis. Relative to Aspergillus fumigatus, colonization of the respiratory tract by multiple strains, and of deep organs by only a single strain were observed. On the other hand, the few studies which focused on other fungi isolated from patients with cystic fibrosis have suggested that colonization occurs primarily by a dominant genotype.

Aspergillus terreus appears to have become an increasingly frequent cause of opportunistic infections in the University Hospital of Innsbruck (UHI) and is of serious concern because of in vivo and in vitro resistance to amphotericin B. In order to determine the possible relationship between environmental contamination by A. terreus and the occurrence of invasive aspergillosis, a 1-year prospective study (2004-2005) was carried out in the UHI. Isolates obtained from air samples of various high-risk settings and those from surveillance cultures of proven and probable aspergillosis (EORTC/MSG criteria) were examined by genotyping. Within 1 year, 34 and 15 A. terreus isolates were collected from the environment and from patients, respectively. Genotypic analysis with rapid amplification of polymorphic DNA (RAPD) PCR and the combination of three different primers (R108, CII, P4) revealed 46 distinct genotypic profiles (types 1-46). No strain similarity was detected among and within the patients and environmental areas, indicating a great genomic diversity in A. terreus, which is common in the environment of Innsbruck and a source of invasive infections in immunosuppressed patients. Genotypical diversity was found in clinical and environmental A. terreus isolates.

The possible co-existence of different genotypes of Aspergillus fumigatus in the same case was studied in five patients colonized or infected by this opportunistic mould. A total of 22 isolates were typed by random amplified polymorphic DNA (RAPD) and microsatellite analysis. Differences in the mating type and invasiveness of the isolates were also considered. The combination of four arbitrary primers used in RAPD typing differentiated all the isolates. In microsatellite analysis, at least two different genotypes were identified in four of the five patients. The 22 isolates showed elastase activity after 10 days of incubation at 37 degrees C in an elastin-containing medium. The presence of strains of compatible mating type was observed in one of the colonized patients and one of the individuals with invasive aspergillosis. Some isolates that belonged to the same genotype in microsatellite analysis were of a different mating type. Taken together, our results suggest that multiple isolates of A. fumigatus obtained from colonized or infected patients may differ not only in their genotypes, but also in their invasiveness and mating types. Furthermore, mating type determination may be of great assistance in differentiating some isolates, as two isolates of different mating type cannot be genotypically identical.

Aspergillus terreus isolates collected at the M. D. Anderson Cancer Center in Houston, Texas, and at the University Hospital of Innsbruck, Austria, were analyzed using random amplification of polymorphic DNA-PCR with three different primers. No strain similarity in either institution was detected, indicating great genetic diversity of A. terreus.

Aspergillus terreus, a less common pathogen, appears to be an emerging cause of infection at our institution, the Medical University Hospital of Innsbruck. Thus the epidemiology and outcome of A. terreus infections over the past 10 years was assessed. We analysed 67 cases of proven invasive aspergillosis (IA) according to the European Organisation for Research and Treatment of Cancer/Mycoses Study Group criteria, investigated antifungal susceptibility of amphotericin B (AMB), voriconazole and caspofungin and performed molecular typing of A. terreus. Patients with proven IA caused by A. terreus (n = 32) and non-A. terreus (n = 35) were evaluated. The two groups were comparable in terms of age, gender, underlying disease, antifungal prophylaxis and duration of neutropenia (P > 0.05). Leukaemia was the most common underlying malignancy. Fungal dissemination occurred in 63% of the patients. Aspergillus terreus infections were associated with a lower response rate to AMB therapy (20%), compared with 47% for patients with non-A. terreus infections (P < 0.05). In vitro, A. terreus was found to be resistant to AMB and molecular typing discriminated between patients isolates, showing a high strain diversity with 26 distinct types (I-XXVI) identified by combination of three primers. Aspergillus terreus infections displayed evidence of AMB resistance in vitro and in vivo and were associated with a high rate of dissemination and poor outcome; A. terreus causes systemic infections of endemic character in Tyrol, Austria. The onset of A. terreus infection depends not on the degree of immunosuppression but on environmental Aspergillus spp. exposure.

Aspergillus terreus is a ubiquitous fungus in our environment. It is an opportunistic human pathogen and economically important as the main producer of lovastatin, a cholesterol lowering drug. Our aim was to examine the genetic variability of A. terreus and closely related species using molecular and analytical techniques. Lovastatin production was examined by HPLC. Lovastatin was produced by seven isolates belonging to the species A. terreus. RAPD analyses were carried out using 25 different random primers. Neighbor-joining analysis of RAPD data (120 characters) resulted in clustering of the A. terreus isolates into distinct groups. Some correlation was observed between lovastatin producing abilities of the isolates and their position on the dendrogram based on RAPD profiles. The internal transcribed spacer region and the 5.8S rRNA gene of A. terreus and related isolates was also sequenced. Phylogenetic analysis of sequence data let us classify the isolates into different clades which mostly correspond to the species Aspergillus terreus, Aspergillus flavipes, Aspergillus niveus, Aspergillus carneus and Aspergillus janus/A. janus var. brevis. Aspergillus allahabadii, A. terreus var. aureus and A. niveus var. indicus belonged to the A. niveus clade, while an Aspergillus isolate previously classified as A. niveus was most closely related to A. flavipes isolates. Aspergillus anthodesmis formed a distinct branch on the tree. Although it was previously suggested based on 28S rDNA sequence data that Aspergillus section Terrei should include A. carneus and A. niveus isolates, phylogenetic analysis of ITS sequences indicate that A. flavipes isolates are more closely related to A. terreus than A. carneus isolates. Our data suggest that sections Terrei and Flavipedes should be merged. However, further loci should be analysed to draw more definite conclusions.

Invasive fungal infections due to Aspergillus species have become a major cause of morbidity and mortality among immunocompromised patients. Aspergillus terreus, a less common pathogen, appears to be an emerging cause of infection at our institution, the University of Alabama hospital in Birmingham. We therefore investigated the epidemiology of A. terreus over the past 6 years by using culture data; antifungal susceptibility testing with amphotericin B, voriconazole, and itraconazole; and molecular typing with random amplification of polymorphic DNA-PCR (RAPD-PCR). During the study period, the percentage of A. terreus isolates relative to those of other Aspergillus species significantly increased, and A. terreus isolates frequently were resistant to amphotericin B. Molecular typing with the RAPD technique was useful in discriminating between patient isolates, which showed much strain diversity. Further surveillance of A. terreus may better define epidemiology and determine whether this organism is becoming more frequent in relation to other Aspergillus species.

BACKGROUND Invasive oral aspergillosis is a rare complication and only little information on the epidemiology of Aspergillus flavus infection is available. We present here the molecular analysis of the epidemiology of invasive stomatitis due to Aspergillus flavus in patients with acute leukemia. METHODS During a 5-year period (1992-1996), six isolates of A. flavus were obtained from leukemic patients with invasive Aspergillus stomatitis. Random amplification of polymorphic DNA (RAPD) with three different PCR primers was carried out to investigate the DNA typing of the isolates. RESULTS The molecular analysis using RAPD revealed that three isolates of A. flavus obtained in 1992 from three patients were of the same type, whereas each of the isolates from the other three patients had a distinct unique band, resulting in four groups of A. flavus. CONCLUSION As the three patients with invasive oral aspergillosis detected in 1992 were infected by a single strain of A. flavus, the strain was suspected to have caused a nosocomial outbreak of invasive oral aspergillosis in the hematology unit.

Manipulation of the genome of the human pathogen Aspergillus fumigatus is not well developed. Approaches and data from related model organisms are being used to develop molecular genetic systems in A. fumigatus; for example, the molecular typing of strains during infection. A genome-sequencing programme has begun and will form the basis for future development.

Little is known of the phospholipid composition of Aspergillus species. The aim of this study was to determine individual phospholipid analogues in Aspergillus. Twenty-nine clinical and environmental isolates from five Aspergillus species were analysed. Fast atom bombardment mass spectrometric data were considered in two ranges, m/z 190-500 and m/z 500-1000, to facilitate the recognition of major fatty acyl groups and phospholipids. Quantitative comparison of major anions in both m/z ranges was undertaken. Confirmation of major phospholipid anions from eight representative isolates was achieved by tandem mass spectrometry. The major phospholipid families were phosphatidic acid (PA), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidylserine (PS). Anions were detected consistent with the presence of specific phospholipid moieties, such as palmitoyl-linolenoyl phosphatidic acid, palmitoyl-oleoylphosphatidylethenolamine, oleoyl-linoleoyl-phophatidylserine and palmitoyl-linoleoyl-phosphatidylinositol. It appears that there is some commonality of sn1 or sn2 fatty acyl substituents, frequently C18:2 at sn1 accompanied by C16:0 at sn2, with differing molecular weights being attributable to analogues with differing head groups. Differences in certain phospholipids (e.g. minor peak with m/z 933) were detected between A. fumigatus, A. nidulans and other species examined which could have diagnostic value.

Azole-resistant oropharyngeal and oesophageal candidiasis is a recent phenomenon observed in patients with AIDS usually previously treated with fluconazole. Some variation has been observed in antifungal susceptibility testing among separate colonies of Candida albicans from the same patient. This raises the question of whether there are multiple clones present or simply phenotypic variation in expression of azole resistance. To address this question we took 18 isolates grown from multiple swabs taken before and after experimental azole therapy from a single HIV-positive individual with fluconazole-resistant oral candidiasis and compared morphotype, karyotype, PCR-based DNA typing and azole susceptibility. Ten of the isolates were from a single 2-day period. Amongst these 10 there were seven morphotypes, five karyotypes and four polymerase chain reaction (PCR) types. Three further morphotypes, one karyotype and two PCR types were found amongst the eight isolates obtained during the subsequent 4 months. Limited variation in susceptibility to two azoles--fluconazole and D0870--was also seen. This work emphasizes both the large genotype and phenotypic variability of C. albicans isolates in the mouth of AIDS patients with fluconazole resistance, and the difficulties in interpretation of present typing methods.

We examined fast atom bombardment mass spectra (FAB-MS) of 29 clinical isolates of Aspergillus, from five pathogenic species, for the presence of phthioic acid anions (m/z 395.6) when grown at 37 degrees C. Phthioic acid was detected in only one of 12 A. fumigatus, three of nine A. terreus and one of four A. niger isolates. Phthioic acid is unlikely to be a major pathogenicity determinant of Aspergillus.

Wound infections with Aspergillus are surprisingly rare given the quantity of spores in hospital air. We report the first case of infection of abdominal viscera due to Aspergillus in a patient with a laparostomy for Crohn's disease. Amphotericin B effected a cure. Sampling of air from the patient's environment yielded one isolated of Aspergillus fumigatus that matched the patient's isolates. Other examples of surgical wounds being contaminated by Aspergillus are reviewed.

Although several typing methods have been described for Shiga-like toxin-producing Escherichia coli O157, the methods are somewhat cumbersome. Using 22 isolates of Escherichia coli O157 and five other Escherichia coli isolates, two primers (M13 core sequence and 970-11) were found to give excellent differentiation between isolates using random amplified polymorphic DNA (RAPD). Using only the presence or absence of variable bands, a matrix of 20 variable characters was identified. From these characters, similarity coefficients were calculated and a phenogram constructed. All of the Escherichia coli O157 isolates were easily distinguished from the non-O157 Escherichia coli isolates. Using a 95% similarity cutoff, we found 13 RAPD types among the 22 Escherichia coli O157 isolates. Isolates thought to be identical by toxin and phage typing as well as by epidemiological association were distinguished, and others thought to be distinct by lack of epidemiological association were identical. RAPD using M13 and 970-11 primers is a potentially useful typing tool for Escherichia coli isolates of serotype O157 and possibly other Escherichia coli isolates.

Extracellular phospholipase activity has been implicated in the pathogenesis of several bacterial infections. Recently, extracellular phospholipase activity has been proposed as a virulence factor in the opportunistic yeast Candida albicans. Aspergillus fumigatus is the most pathogenic member of its genus, responsible for > 90% of infections. Previously, no specific virulence factors have been determined. We investigated the ability of A. fumigatus to produce extracellular phospholipases at 37 degrees C. Fast atom bombardment was used to compare lipid-containing media before and at 5-h intervals during shaking culture of A. fumigatus. Lipids were extracted and analyzed. Many anions corresponding to phospholipid breakdown products were identified. Specific anion species identified indicated phospholipase A, B, C (PLC), and D activities. PLC activity was further investigated by using the synthetic substrate p-nitrophenylphosphorylcholine. PLC activity was initially observed after 30 h of growth and accumulated in broth cultures up to 50 h. At 55 h, there was a sharp increase in PLC activity which coincided with cultures reaching the stationary phase. Activity of the PLC was measured at different temperatures, with greater activity occurring at 37 degrees C than at lower temperatures. Phospholipases could represent a virulence determinant in A. fumigatus.

Invasive aspergillosis is often nosocomially acquired and carries a high mortality. Molecular typing methods to discriminate isolates have now been developed. Using simple restriction endonuclease (Sal1 and Xho1) digestion of total genomic DNA, we have typed 25 epidemiologically-related isolates of A. fumigatus from six hospital episodes of invasive aspergillosis. Eight DNA types were found and in each case the DNA type matched precisely the epidemiological data. Thus DNA typing of A. fumigatus can provide the means to match isolates from linked sources and distinguish isolates from diverse origins.

In the last four years, several molecular typing methods have been applied to various Aspergillus species, primarily A. fumigatus and A. niger. Many 'clones' or DNA types exist in the environment and in patients. The most discriminatory methods appear to be restriction fragment length polymorphism analysis with or without specific probes and random amplification of polymorphic DNA analysis. Insufficient work on interlaboratory reproducibility has been done. Most patients are infected by single DNA types, although occasionally multiple isolates are detected. Little work has been done with regard to detailed hospital epidemiology using molecular typing methods.

OBJECTIVES: Resistance to azole antifungal drugs in Aspergillus fumigatus that is not mediated by target gene mutations is now common in some locations. The aim of this study was to investigate possible new mechanisms of resistance in non-target resistance. METHODS: Twelve azole-resistant A. fumigatus isolates previously shown not to carry mutations in cyp51A were tested to determine whether the alternative cyp51B gene was overexpressed. RESULTS: Of 12 isolates one showed overinduction of cyp51B after exposure to itraconazole and another showed high constitutive expression of cyp51B. CONCLUSIONS: cyp51B overexpression is a possible azole resistance mechanism in A. fumigatus.

A simple HPLC method has been developed to measure imatinib and N-desmethylimatinib (norimatinib) in plasma or serum at concentrations attained during therapy. Adaptation of this method to LC-MS/MS also allows dasatinib assay. A small sample volume (100 μL HPLC-UV, 50 μL LC-MS/MS) is required and analysis time is <5 min in each case. Detection was by UV (270 nm) or selective reaction monitoring (two transitions per analyte) tandem mass spectrometry. Assay calibration was linear (0.05-10 mg/L imatinib, 0.01-2.0 mg/L norimatinib and 1-200 µg/L dasatinib), with acceptable accuracy (86-114%) and precision (<14% RSD) for both methods. A comparison between whole blood and plasma confirmed that plasma is the preferred sample for imatinib and norimatinib assay. For dasatinib, although whole blood concentrations were slightly higher, plasma is still the preferred sample. Despite considerable variation in the (median, range) plasma imatinib and norimatinib concentrations in patient samples [1.66 (0.02-4.96) and 0.32 (0.01-0.99) mg/L, respectively, N = 104], plasma imatinib was >1 mg/L (suggested target for response) in all but one sample from patients achieving complete molecular response. As to dasatinib, the median (range) plasma dasatinib concentration was 13 (2-143) µg/L (N = 33). More observations are needed to properly assess the potential role of therapeutic drug monitoring in guiding treatment with dasatinib. Copyright © 2012 John Wiley & Sons, Ltd.

In this paper we reviewed the latest literature on molecular techniques used in diagnosis and epidemiology of infections caused by pathogenic fungi. Traditional methods used for the identification and typing of medically relevant fungi include morphological and biochemical analysis. These methods are time-consuming and base on phenotypic features what makes them unreliable. We described the usefulness in mycological studies of fast and very sensitive molecular methods which rely on PCR and hybridization techniques.

Numerous patients were diagnosed with aspergillosis in a nosocomial outbreak caused by Aspergillus fumigatus and Aspergillus flavus. Thirty-three isolates of the former and 28 isolates of the latter were collected from the hospital environment and from the patients and studied for genetic relatedness by random amplified microsatellites (RAMS) analysis, in which two polymorphic regions were tested. Twenty-eight genotypes of A. fumigatus and 23 genotypes of A. flavus were identified. Four patients were infected by two isolates with the same genotype as the environmental isolates. One clinical genotype was shared by three patients and another was shared by two patients. We found that RAMS was useful for fingerprinting Aspergillus spp.

Aspergillus terreus is a ubiquitous fungus in our environment. It is an opportunistic human pathogen and economically important as the main producer of lovastatin, a cholesterol lowering drug. Our aim was to examine the genetic variability of A. terreus and closely related species using molecular and analytical techniques. Lovastatin production was examined by HPLC. Lovastatin was produced by seven isolates belonging to the species A. terreus. RAPD analyses were carried out using 25 different random primers. Neighbor-joining analysis of RAPD data (120 characters) resulted in clustering of the A. terreus isolates into distinct groups. Some correlation was observed between lovastatin producing abilities of the isolates and their position on the dendrogram based on RAPD profiles. The internal transcribed spacer region and the 5.8S rRNA gene of A. terreus and related isolates was also sequenced. Phylogenetic analysis of sequence data let us classify the isolates into different clades which mostly correspond to the species Aspergillus terreus, Aspergillus flavipes, Aspergillus niveus, Aspergillus carneus and Aspergillus janus/A. janus var. brevis. Aspergillus allahabadii, A. terreus var. aureus and A. niveus var. indicus belonged to the A. niveus clade, while an Aspergillus isolate previously classified as A. niveus was most closely related to A. flavipes isolates. Aspergillus anthodesmis formed a distinct branch on the tree. Although it was previously suggested based on 28S rDNA sequence data that Aspergillus section Terrei should include A. carneus and A. niveus isolates, phylogenetic analysis of ITS sequences indicate that A. flavipes isolates are more closely related to A. terreus than A. carneus isolates. Our data suggest that sections Terrei and Flavipedes should be merged. However, further loci should be analysed to draw more definite conclusions.

Ochratoxin A (OTA) is a carcinogenic and nephrotoxic mycotoxin that has been detected in a variety of food products, including green coffee beans. About 80% of Aspergillus carbonarius strains collected from coffee beans are able to produce OTA on this substrate. The rapid identification of this fungal species would be desirable. RAPD assays were applied to identify amplification products specific for A. carbonarius. One selected fragment, denoted OPX7809, was cloned and sequenced. Based on the nucleotide sequence obtained, specific oligonucleotides (OPX7F809 and OPX7R809) were designed and used as primers for DNA amplification. One amplified band of 809 bp was obtained from A. carbonarius genomic DNA, whereas no amplified fragment from DNA of other Aspergillus species was detected. This PCR analysis was also successfully employed to detect A. carbonarius in coffee beans. This PCR assay could contribute to the early and rapid detection of the potential presence of OTA in coffee samples.

We developed a PCR-based assay to differentiate medically important species of Aspergillus from one another and from other opportunistic molds and yeasts by employing universal, fungus-specific primers and DNA probes in an enzyme immunoassay format (PCR-EIA). Oligonucleotide probes, directed to the internal transcribed spacer 2 region of ribosomal DNA from Aspergillus flavus, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus terreus, Aspergillus ustus, and Aspergillus versicolor, differentiated 41 isolates (3 to 9 each of the respective species; P < 0.001) in a PCR-EIA detection matrix and gave no false-positive reactions with 33 species of Acremonium, Exophiala, Candida, Fusarium, Mucor, Paecilomyces, Penicillium, Rhizopus, Scedosporium, Sporothrix, or other aspergilli tested. A single DNA probe to detect all seven of the most medically important Aspergillus species (A. flavus, A. fumigatus, A. nidulans, A. niger, A. terreus, A. ustus, and A. versicolor) was also designed. Identification of Aspergillus species was accomplished within a single day by the PCR-EIA, and as little as 0.5 pg of fungal DNA could be detected by this system. In addition, fungal DNA extracted from tissues of experimentally infected rabbits was successfully amplified and identified using the PCR-EIA system. This method is simple, rapid, and sensitive for the identification of medically important Aspergillus species and for their differentiation from other opportunistic fungi.

Candida parapsilosis was detected in environmental swabs and batches of total parenteral nutrition (TPN) products after routine monitoring. The isolates were analysed using randomly amplified polymorphic DNA (RAPD) to determine clonality and establish the most likely source of contamination. Of 20 isolates analysed, 18 were indistinguishable clonally and were found to be associated with particular work stations. The application of regular testing using a system such as the BacT/Alert, and molecular studies for epidemiological analysis, is of benefit to producers of medical products such as TPN to ensure patient safety.

Invasive fungal infections due to Aspergillus species have become a major cause of morbidity and mortality among immunocompromised patients. Aspergillus terreus, a less common pathogen, appears to be an emerging cause of infection at our institution, the University of Alabama hospital in Birmingham. We therefore investigated the epidemiology of A. terreus over the past 6 years by using culture data; antifungal susceptibility testing with amphotericin B, voriconazole, and itraconazole; and molecular typing with random amplification of polymorphic DNA-PCR (RAPD-PCR). During the study period, the percentage of A. terreus isolates relative to those of other Aspergillus species significantly increased, and A. terreus isolates frequently were resistant to amphotericin B. Molecular typing with the RAPD technique was useful in discriminating between patient isolates, which showed much strain diversity. Further surveillance of A. terreus may better define epidemiology and determine whether this organism is becoming more frequent in relation to other Aspergillus species.

Identification and typing of fungal isolates is a prerequisite for control and prevention of nosocomial infections. As the discriminatory power of phenotypic methods is not sufficient for epidemiological purposes, genotyping methods such as DNA fingerprinting, random amplification of polymorphic DNA (RAPD) analysis, or pulsed field gel electrophoresis (PFGE) are preferred. To our knowledge, this study is the first application of PFGE for typing Rhodotorula mucilaginosa strains. The PFGE patterns of six clinical isolates produced two different karyotypes, which were confirmed by RAPD analysis. Five strains isolated from bloodstream infections from three different institutions showed the same karyotype and RAPD patterns, while the urological specimen differed slightly.