Aspergillus Fumigatus Isolates Research Articles

Genetic diversity ofAspergillus fumigatusin indoor hospital environments

Environmental isolates of Aspergillus fumigatus are less studied than those recovered from clinical sources. In the present study, the genetic diversity among such environmental isolates was assessed, as well as their dispersion ability and the acquisition of new strains in 19 medical units of the same hospital. A. fumigatus isolates were genotyped using a single multiplex PCR-based reaction with eight microsatellite markers and an insertion/deletion polymorphism. A total of 130 unique genotypes were found among a total of 250 A. fumigatus isolates. Genotypic diversity ranged from 0.86 to 1 in samples from hospital rooms, and there was no correlation between these samples and the presence of high-efficiency particulate air filters or any other air filtration system. Four of the six most prevalent A. fumigatus strains were recovered from water samples. The occurrence of microvariation was common among environmental isolates, which affected each of the microsatellite markers. The assessment of the genetic diversity of A. fumigatus is a useful tool for illustrating the presence or absence of specific clonal populations in a clinical setting. A. fumigatus populations were highly dynamic indoors, and new populations were found in just a few months. Due to the high indoor dispersion capability of A. fumigatus, more attention should be given to strains with increased pathogenic potential or reduced susceptibility to anti-fungal drugs.

Development of Azole Resistance in Aspergillus fumigatus during Azole Therapy Associated with Change in Virulence

Four sequential Aspergillus fumigatus isolates from a patient with chronic granulomatous disease (CGD) eventually failing azole-echinocandin combination therapy were investigated. The first two isolates (1 and 2) were susceptible to antifungal azoles, but increased itraconazole, voriconazole and posaconazole MICs were found for the last two isolates (3 and 4). Microsatellite typing showed that the 4 isolates were isogenic, suggesting that resistance had been acquired during azole treatment of the patient. An immunocompromised mouse model confirmed that the in vitro resistance corresponded with treatment failure. Mice challenged with the resistant isolate 4 failed to respond to posaconazole therapy, while those infected by susceptible isolate 2 responded. Posaconazole-anidulafungin combination therapy was effective in mice challenged with isolate 4. No mutations were found in the Cyp51A gene of the four isolates. However, expression experiments of the Cyp51A showed that the expression was increased in the resistant isolates, compared to the azole-susceptible isolates. The microscopic morphology of the four isolates was similar, but a clear alteration in radial growth and a significantly reduced growth rate of the resistant isolates on solid and in broth medium was observed compared to isolates 1 and 2 and to unrelated wild-type controls. In the mouse model the virulence of isolates 3 and 4 was reduced compared to the susceptible ones and to wild-type controls. For the first time, the acquisition of azole resistance despite azole-echinocandin combination therapy is described in a CGD patient and the resistance demonstrated to be directly associated with significant change of virulence.

Potential synergistic activity of antimycotic substances in combination with human platelets against Aspergillus fumigatus

Sir, Invasive aspergillosis is increasingly recognized in immunocompromised hosts. Patients with prolonged and deep granulocytopenia following chemotherapy for haemato-oncological disorders or allogeneic bone marrow transplant recipients are particularly at risk. Platelets are second in abundance to red blood cells in the circulation, with 1.5–4.0×10 platelets/mL of blood in healthy individuals. They are essential components in haemostasis, but they also play an important role in antimicrobial host defence. Recently, we observed that platelets released serotonin after contact with aspergilli. Furthermore, platelets attenuated the virulence of Aspergillus spp. as the cell wall compound galactomannan was significantly decreased. Several in vitro studies have indicated that immune effector cells in combination with antifungals exert synergistic effects on aspergilli. However, no data are available on the effects of platelets and antimycotics on aspergilli. Two clinical Aspergillus fumigatus isolates and four antifungal agents were studied: amphotericin B (Sigma–Aldrich, Vienna, Austria); voriconazole (kindly provided by Pfizer, Vienna, Austria); posaconazole (kindly provided by Schering-Plough Research Institute, Kenilworth, NJ, USA); and caspofungin (MSD, Vienna, Austria). The MICs of all drugs used in this study were 0.5 mg/L for both isolates. Based on the MICs, drug concentrations of 0.5 and 1 mg/L were used for the platelet/drug studies. Fresh platelets were provided from the local Department of Immunology and Blood Transfusion. Platelets were collected from healthy donors by thrombocytopheresis using an Amicus cell separator (Baxter, Vienna, Austria). One hundred microlitres each of platelets (1×10/mL) and conidia (1×10/mL) in an effector/target ratio of 100:1 and specific concentrations of antifungals (0.5 and 1 mg/L) were inoculated into microwell plates (Greiner, Vienna, Austria) and incubated at 378C. The morphology of the A. fumigatus strains treated either with the combination of platelets plus antifungals or with each substance or platelets alone was investigated by assessing the germination rate and hyphal elongation, as described previously. Hyphal damage was assessed by a colorimetric assay with the dye 2,3-bis[2-methoxy-4-nitro-5-sulphophenyl]-2-H-tetrazolium-5carboxynilide sodium salt (XTT; Sigma–Aldrich) plus 40 mg/mL coenzyme Q (Sigma–Aldrich) and antifungal activity was calculated as the percentage of hyphal damage. Each experiment was performed with platelets from one donor using duplicate or quadruplicate wells for each condition and strain, and repeated six times. Differences between mean values of both strains were statistically evaluated by repeated-measures analysis of variance followed by Dunnett’s correction for multiple comparisons. A twosided P value of ,0.05 indicated statistical significance. Human platelets plus amphotericin B achieved significantly greater inhibition of the germination rate (P,0.05) than did amphotericin B or platelets alone (Figure 1a). The effect was additive. These results were found with both tested amphotericin B concentrations, although 1 mg/L revealed a better inhibitory effect than the lower concentration (Figure 1a). With caspofungin, the germination rate was not significantly reduced (Figure 1b). When azoles were used in combination with platelets, the inhibitory effect was found to be additive in comparison with either azole or platelets alone (Figure 1c and d). As found for the germination rate, human platelets plus amphotericin B achieved significantly greater inhibition of hyphal elongation (P,0.05) than amphotericin B or platelets alone. Hyphal elongation was significantly reduced in all tested aspergilli, either under platelet or caspofungin treatment. When used in combination, the effect was found to be additive. The combination of platelets plus either azole at any concentration tested did not significantly enhance the reduction of hyphal elongation. Platelets decreased the ability of hyphae to reduce XTT; however, the combination of platelets plus antimycotics had no additive effect on hyphal damage (data not shown). Our findings indicate that platelets in combination with antimycotics exert additive effects in reducing the germination rate and hyphal elongation of A. fumigatus in vitro. Among the tested antimycotic substances, amphotericin B revealed the best results in combination with human platelets. However, platelets plus antimycotics were not additive for hyphal damage. In the immunocompromised patient, inhaled Aspergillus conidia germinate into hyphae, the growing and invading structures of filamentous fungi. Consequently, blocking fungal germination and delaying hyphal growth is crucial in preventing invasive disease. In our study, the combination of platelets plus amphotericin B synergistically enhanced the antifungal activity (P,0.05) in reducing germination rate and hyphal elongation. Amphotericin B is known to complex with sterols in the fungal cell membrane, leading to pore formation and

In vitro antifungal activity of aminosterols against moulds isolated from cystic fibrosis patients

Sir, We have recently demonstrated that aminosterol derivatives (ASDs) possess an interesting in vitro antibacterial activity against multidrug-resistant bacteria recovered from cystic fibrosis (CF) patients. However, mould infections are also a serious threat for CF patients. Indeed, the fragile pulmonary environment of these patients facilitates the development of bacterial and fungal infections. Repeated bacterial infections, intensive antibiotic therapy and lung transplantation are considered as risk factors for airway infection and/or colonization by moulds in CF patients. Major problems are related to Aspergillus spp. and Scedosporium spp. that may cause chronic disorders such as asthma, bronchitis and aspergilloma. Amphotericin B, caspofungin and azoles are currently used in the management of fungal infections. However, in addition to acquired resistance resulting from antifungal treatment pressure as noted for Aspergillus spp., intrinsic resistance to available antifungals reported in some fungi such as Fusarium, Rhizopus, Rhizomucor and Scedosporium spp. represents a major issue, highlighting the need to develop new antifungal compounds. ASDs have been evaluated only against reference fungal strains. We evaluated herein for the first time the in vitro antifungal activity of squalamine and one of our compounds (ASD 1; the chemical structure is available in Alhanout et al.) possessing an in vitro antibacterial effect, against a panel of clinical mould isolates recovered from sputa of CF patients. All tested isolates were identified at the species level using partial internal transcribed spacer PCR amplification, and the sequencing procedure and sequences have been deposited in GenBank under accession numbers GU594733– GU594779. The in vitro antifungal activities of currently available systemic antifungal drugs were evaluated for comparison purposes. MICs of all tested compounds were determined by the reference broth dilution method as previously reported, and found to be reproducible and in agreement with previous reports for standard antifungal agents as noted using reference fungal strains including four yeasts and one mould (Aspergillus brasiliensis ATCC 16404) (Table 1). Squalamine and ASD 1 displayed MICs for all tested isolates ranging from 8 to 16 mg/L and from 2 to 4 mg/L, respectively. Since no internationally agreed antifungal resistance breakpoints are currently available, we adopted breakpoints proposed in a recent work suggesting MICs .2 mg/L for azoles to designate resistance in Aspergillus spp. For simplicity, we used this MIC to assign resistance to other antifungal drugs for all fungal isolates. In keeping with previous studies, Aspergillus fumigatus, Aspergillus niger and Aspergillus flavus isolates were susceptible to all tested antifungals. Conversely, Aspergillus terreus isolates were resistant to amphotericin B as previously reported and, surprisingly, to itraconazole for our CF clinical isolates, which is unusual. As has been documented for other Aspergillus spp., the resistance of A. terreus to itraconazole probably may be acquired since this fungus chronically colonizes the airways of CF patients that are exposed to various antimicrobial treatments. According to previous reports, Aspergillus ustus was resistant to itraconazole, voriconazole and caspofungin. Resistance to caspofungin and voriconazole was noted for Emericella quadrilineata. Antifungal resistance in Penicillium and Cladosporium spp. was previously reported. Accordingly, two out of four Penicillium griseofulvum isolates were resistant to voriconazole and caspofungin while the other two isolates were resistant to all tested available antifungals. Moreover, Cladosporium spp. were resistant to all antifungals except amphotericin B, while Alternaria spp. were susceptible to all tested antifungals. In accordance with previous findings, resistance to all antifungals was found for all tested isolates of Fusarium proliferatum, Scedosporium prolificans, Pseudallescheria boydii, Scedosporium apiospermum, Rhizopus oryzae and Rhizomucor tauricus. Hence, airway colonization with these moulds is quite worrying, calling for an effective therapy to be available. Conversely, tested aminosterols and particularly ASD 1 demonstrated a homogeneous in vitro activity against all susceptible and resistant isolates. This finding suggests that novel mechanistic aspects, distinct from those of available drugs, underlie the antifungal activity of ASDs. While it is known that squalamine acts by disrupting bacterial membranes, the antifungal mechanism of action of ASDs remains unknown. Squalamine and ASD 1 previously demonstrated comparable activities against various bacterial isolates, whilst the present data interestingly show that ASD 1 was superior to squalamine, indicating a probable different effect due to a different chemical structure of the two tested compounds. While the safety of squalamine has been demonstrated in clinical studies, no in vitro or in vivo toxicity studies have been reported for synthesized aminosterols. Nevertheless, since ASDs were reported to act as membrane disruptors, such a wide antimicrobial spectrum may encompass a potential toxicity that should be further investigated. Overall, we have demonstrated herein that ASDs possess an interesting in vitro antifungal activity, advocating their development for local administration, for example as aerosols especially in the context of CF. Further studies are warranted in order to evaluate their in vivo antimicrobial activities in animal models.

Interstrain variability in the virulence ofAspergillus fumigatusandAspergillus terreusin aToll-deficientDrosophilafly model of invasive aspergillosis

Members of the genus Aspergillus are opportunistic fungal pathogens characterized by their genomic diversity. However, whether variations among Aspergillus strains and species at the genome level translate into significant differences in virulence is unclear. Therefore, we studied the interstrain and interspecies variations in virulence for a collection of Aspergillus fumigatus and Aspergillus terreus isolates using a previously described model of invasive aspergillosis in Toll-deficient fruit flies. We then looked for associations between survival in the fly model and strain relatedness as defined by repetitive-sequence polymerase chain reaction (rep-PCR). We observed no significant differences in the survival of flies infected with A. fumigatus vs. A. terreus or flies infected with colonizing vs. invasive isolates of either species. However, in both Aspergillus species we observed significant interstrain variability in fly survival (P<0.001 by the log-rank test). Using rep-PCR, we identified two dominant A. fumigatus clades that were associated with significantly different survival rates in Toll-deficient flies (P=0.007). We conclude that the fly model of invasive aspergillosis enables high-throughput screening of Aspergillus species for variations in virulence and may uncover distinct A. fumigatus clades that differ in their pathogenicity.

Use of a High-Resolution Melt Assay To Characterize Codon 54 of the cyp51A Gene of Aspergillus fumigatus on a Rotor-Gene 6000 Instrument

A high-resolution melt (HRM) assay using a Rotor-Gene 6000 instrument was developed to characterize the codon for glycine 54 in the cyp51A genes from 13 reference isolates and 12 clinical isolates of Aspergillus fumigatus. Mutations in this codon confer reduced susceptibility to itraconazole and posaconazole. The assay is simple to perform, and a result of "wild type" or "mutant" is available after approximately 1 h following DNA extraction using commercially available reagents and conventional primers.

Novel mixed-format real-time PCR assay to detect mutations conferring resistance to triazoles in Aspergillus fumigatus and prevalence of multi-triazole resistance among clinical isolates in the Netherlands

The aim of this study was: (i) to study the prevalence of triazole-resistant Aspergillus fumigatus isolates in the Netherlands; and (ii) to design rapid real-time PCR methods to identify such isolates. A novel mixed-format real-time PCR assay is described for the detection of mutations leading to triazole resistance in A. fumigatus. One set of PCR primers and a probe carrying a single fluorescent label in combination with a double-stranded DNA fluorescent dye allow simultaneous detection of (a) specific mutation(s) as well as of the amplified product that serves as an internal amplification control. The method was applied to a random collection of 209 clinical isolates from throughout the Netherlands and was compared with phenotypic susceptibility testing. A total of four triazole-resistant isolates were identified, resulting in a prevalence of resistant isolates of <2%. All four isolates contained an identical combination of mutations leading to multi-triazole resistance, as reported before by others. Molecular testing results were 100% concordant with phenotypic susceptibility testing. Although in specific patient populations the prevalence of resistance in A. fumigatus may be an emerging problem, in the general population it is still relatively low. The novel real-time PCR format allows rapid and reliable identification of such isolates.

Microsatellite genotyping and virulence assessment of Aspergillus fumigatus isolates from white stork nestlings and their environment

Microsatellite genotyping and virulence assessment of Aspergillus fumigatus isolates from white stork nestlings and their environment

The Antifungal Activity of Sarcococca saligna Ethanol Extract and its Combination Effect with Fluconazole against Different Resistant Aspergillus Species

Microbial resistance is a major drawback in chemotherapy of microbial or fungal infection disease. In this study, the antifungal activity of ethanol extract of a selected plant (Sarcococca saligna) has been investigated against clinical isolates of Aspergillus niger, Aspergillus treus, Aspergillus flavus, and Aspergillus fumigatus. Also, the enhancement of the antifungal activity of fluconazole by this extract was further evaluated against mentioned test strains. Conventional disk diffusion method was used to assay the antifungal activity of S. saligna ethanol extract in the absence and presence of fluconazole. The highest antifungal activity was observed against A. treus. The ethanol extract of S. saligna enhanced the antifungal activity of fluconazole against A. niger and A. treus and A. flavus. At the highest tested contents (4 mg/disk), 1.15-, 0.64-, and 2.47-fold increases in inhibition zone surface area were observed for A. niger, A. treus, and A. flavus, respectively. However, no enhancing effect was observed for this plant extract against Aspergillus fumigates at tested contents (0.5, 1, 2, 3, and 4 mg/disk). In a separate experiment, the general cytotoxicity of the ethanol extract of S. saligna was examined with brine shrimp assay. This plant extract showed low cytotoxicity against Artemia salina (LC(50) = 186 microg/ml).

Identification of novel cell wall destabilizing antifungal compounds using a conditional Aspergillus nidulans protein kinase C mutant

Despite the need for novel drugs to combat fungal infections, antifungal drug discovery is currently limited by both the availability of suitable drug targets and assays to screen corresponding targets. The aim of this study was to screen a library of small chemical compounds to identify cell wall inhibitors using a conditional protein kinase C (PKC)-expressing strain of Aspergillus nidulans. This mutant is specifically susceptible to cell wall damaging compounds under PKC-repressive growth conditions. The inhibitory effect of a library of small chemical compounds was examined in vitro using the conditional A. nidulans PKC strain and a panel of pathogenic fungal isolates. Microscopy was used to assess alterations to fungal ultrastructure following treatment. Three 'hit' compounds affecting cell wall integrity were identified from a screen of 5000 small chemical compounds. The most potent compound, CW-11, was further characterized and shown to specifically affect cell wall integrity. In clinical isolates of Aspergillus fumigatus, CW-11 induces morphological changes characteristic of damage to the cell wall, including wall thickening and rupturing. Analysis of the susceptibility of A. fumigatus and A. nidulans cell wall and signalling pathway mutants to CW-11 suggests that its mode of action differs from that of the antifungals caspofungin and voriconazole. This work demonstrates the feasibility of using a conditional Aspergillus mutant to conduct a small-molecule library screen to identify novel 'hit' compounds affecting cell wall integrity.

Summary Isolation and diagnosis of some fungal types from external ear canal infection cases with sensitivity test to some antifungal agents

This research included isolation of some types of fungi of molds and yeasts from cases of inflammation ofexternal ear canal in human from AL- yarmouk Teaching Hospital, by collection of 50 samples from males andfemales and from different ages during summer season , also 10 samples from non infected cases were taken ascontrol. Also sensitivity test of some isolates against some important antifungal agents which commonly usedin the treatments of infection of external ear canale cases.After cultivation of isolates on saboroud dextrose agar, diagnosed as follows :9 isolates of Aspergillus niger, 6 isolates of Aspergillus fumigatus, 3 isolates of Aspergillus flavus, one isolate ofAspergillus terrus, 10 isolates of Candida albicans, 5 isolates of Candida tropicalis, 2 isolates of Candidaparapsilosis.The results of the sensitivity tests of some isolates against the antifungal agents appeared as follows: the isolatesof Aspergillus spp. were very sensitive to Clotrimazole and Econazole, while were less sensitive againstNystatine.Whereas the species of Candida were very sensitive to Nystatine and Econazole, but less sensitive againstClotrimazole.

Utility of a proposed CSP typing nomenclature for Australian Aspergillus fumigatus isolates: Identification of additional CSP types and suggested modifications

A recently developed CSP typing scheme and proposed nomenclature was applied to a collection of 164 clinical and environmental Aspergillus fumigatus isolates from Melbourne, Australia. Fifteen CSP variants were observed overall, including three that were not reported in the original nomenclature that described 19 CSP variants, raising the possibility of phylogeographic differences between the Australian and the previously studied European and North American A. fumigatus populations. However, those CSP variants that were common between this and the previous studies appeared to have a broadly similar prevalence. The presence of an additional CCT codon in the 3′ flanking region of some CSP variants was also observed in homologous Neosartorya fischeri sequence, suggesting that the absence of this codon in other isolates is due to codon deletion, rather than its presence representing a duplication. We recommend a number of modifications to the proposed CSP type nomenclature to accommodate these new findings.

Population structure and diversity of the pathogenic fungus Aspergillus fumigatus isolated from different sources and geographic origins

Fifty-five clinical and environmental Aspergillus fumigatus isolates from Mexico, Argentina, France and Peru were analyzed to determine their genetic variability, reproductive system and level of differentiation using amplified fragment length polymorphism markers. The level of genetic variability was assessed by measuring the percentage of polymorphic loci, number of effective alleles, expected heterozygocity and by performing an association index test (I(A)). The degree of genetic differentiation and variation was determined using analysis of molecular variance at three levels. Using the paired genetic distances, a dendrogram was built to detect the genetic relationship among alleles. Finally, a network of haplotypes was constructed to determine the geographic relationship among them. The results indicate that the clinical isolates have greater genetic variability than the environmental isolates. The I(A) of the clinical and environmental isolates suggests a recombining population structure. The genetic differentiation among isolates and the dendrogram suggest that the groups of isolates are different. The network of haplotypes demonstrates that the majority of the isolates are grouped according to geographic origin.

Utility of CSP typing to sub-type clinical Aspergillus fumigatus isolates and proposal for a new CSP type nomenclature

CSP typing is a newly developed sub-typing strategy that employs comparative DNA sequence analysis of the 12-mer tandem repeat region of the AFUA_3G08890 gene. In order to allow standardization of analysis and exchange of results between laboratories, we propose a new nomenclature for individual CSP repeats as well as for CSP types. A collection of 209 clinical isolates of Aspergillus fumigatus recovered from various hospitals throughout The Netherlands was analyzed by using CSP typing and this newly proposed nomenclature. Eighteen different CSP types were recognized, positioning the CSP gene as a typing target between the relatively low discriminatory MLST loci and the highly discriminatory microsatellite markers. CSP typing may be a welcome addition to the existing molecular methods to study the diversity of A. fumigatus at the sub-population level. The results also show the presence of lineages of closely related CSP types within the A. fumigatus population, adding unique and valuable information about the population structure of A. fumigatus.

Phenotypic and genotypic characterization of Aspergillus lentulus and Aspergillus fumigatus isolates in a patient with probable invasive aspergillosis

Characteristic morphological and phylogenetic analyses demonstrated the presence of Aspergillus fumigatus and Aspergillus lentulus as the aetiological agents in a case of probable invasive aspergillosis (IA). This is believed to be the first report of an A. lentulus strain isolated from a patient with probable IA in Argentina.

Development and Validation of a Microsphere-Based Luminex Assay for Rapid Identification of Clinically Relevant Aspergilli

A Luminex-based assay for the rapid identification of Aspergillus species was designed, optimized, and validated with 131 clinical isolates of Aspergillus fumigatus, A. flavus, A. niger, A. terreus, A. ustus, and A. versicolor. The six species-specific probes were directed toward the internal transcribed spacer 1 (ITS-1) region and tested in a multiplex format with results generated within 6 h. Species identifications generated by the Aspergillus Luminex assay were 100% concordant with results from comparative sequence analyses of the ITS-1 region and showed excellent specificity. The Aspergillus Luminex assay is a rapid, relatively simple method that may prove to be a useful diagnostic tool for rapid Aspergillus identification in clinical laboratory settings.

Interstrain variability in the virulence of Aspergillus fumigatus and Aspergillus terreus in a Toll-deficient Drosophila fly model of invasive aspergillosis

Members of the genus Aspergillus are opportunistic fungal pathogens characterized by their genomic diversity. However, whether variations among Aspergillus strains and species at the genome level translate into significant differences in virulence is unclear. Therefore, we studied the interstrain and interspecies variations in virulence for a collection of Aspergillus fumigatus and Aspergillus terreus isolates using a previously described model of invasive aspergillosis in Toll-deficient fruit flies. We then looked for associations between survival in the fly model and strain relatedness as defined by repetitive-sequence polymerase chain reaction (rep-PCR). We observed no significant differences in the survival of flies infected with A. fumigatus vs. A. terreus or flies infected with colonizing vs. invasive isolates of either species. However, in both Aspergillus species we observed significant interstrain variability in fly survival (P<0.001 by the log-rank test). Using rep-PCR, we identified two dominant A. fumigatus clades that were associated with significantly different survival rates in Toll-deficient flies (P=0.007). We conclude that the fly model of invasive aspergillosis enables high-throughput screening of Aspergillus species for variations in virulence and may uncover distinct A. fumigatus clades that differ in their pathogenicity.

MLST versus microsatellites for typingAspergillus fumigatusisolates

In recent years, there has been a clear and growing tendency to use exact typing methods for discrimination between microbial isolates. Exact typing methods that yield an unambiguous typing result offer a number of advantages over conventional methods in the generation of typing data that is reproducible, portable and exchangeable. Two such methods are multi-locus sequence typing (MLST) and microsatellite-based typing. Here I will discuss the basic principles of both methods and compare them from a practical and performance point of view with respect to typing Aspergillus fumigatus isolates. Microsatellites offer the best available typing option by outperforming MLST in terms of speed, throughput, costs and discriminatory power. This latter advantage of microsatellites is a direct consequence of their inherent instability. This (in)stability of individual microsatellite markers and alleles should be taken into account in the interpretation of microsatellite-based typing data.

Emergence of Azole Resistance in Aspergillus fumigatus and Spread of a Single Resistance Mechanism

BackgroundResistance to triazoles was recently reported in Aspergillus fumigatus isolates cultured from patients with invasive aspergillosis. The prevalence of azole resistance in A. fumigatus is unknown. We investigated the prevalence and spread of azole resistance using our culture collection that contained A. fumigatus isolates collected between 1994 and 2007.Methods and FindingsWe investigated the prevalence of itraconazole (ITZ) resistance in 1,912 clinical A. fumigatus isolates collected from 1,219 patients in our University Medical Centre over a 14-y period. The spread of resistance was investigated by analyzing 147 A. fumigatus isolates from 101 patients, from 28 other medical centres in The Netherlands and 317 isolates from six other countries. The isolates were characterized using phenotypic and molecular methods. The electronic patient files were used to determine the underlying conditions of the patients and the presence of invasive aspergillosis. ITZ-resistant isolates were found in 32 of 1,219 patients. All cases were observed after 1999 with an annual prevalence of 1.7% to 6%. The ITZ-resistant isolates also showed elevated minimum inhibitory concentrations of voriconazole, ravuconazole, and posaconazole. A substitution of leucine 98 for histidine in the cyp51A gene, together with two copies of a 34-bp sequence in tandem in the gene promoter (TR/L98H), was found to be the dominant resistance mechanism. Microsatellite analysis indicated that the ITZ-resistant isolates were genetically distinct but clustered. The ITZ-sensitive isolates were not more likely to be responsible for invasive aspergillosis than the ITZ-resistant isolates. ITZ resistance was found in isolates from 13 patients (12.8%) from nine other medical centres in The Netherlands, of which 69% harboured the TR/L98H substitution, and in six isolates originating from four other countries.ConclusionsAzole resistance has emerged in A. fumigatus and might be more prevalent than currently acknowledged. The presence of a dominant resistance mechanism in clinical isolates suggests that isolates with this mechanism are spreading in our environment.

Investigation of extracellular elastase, acid proteinase and phospholipase activities as putative virulence factors in clinical isolates of Aspergillus species

We investigated the production of extracellular elastase, acid proteinase and phospholipase enzyme activities in clinical isolates of Aspergillus fumigatus, Aspergillus flavus and Aspergillus niger and any possible correlation between the existence of these enzymes and development of invasive aspergillosis. A total of 73 strains (45 A. fumigatus, 23 A. flavus, 5 A. niger) isolated from patients with invasive aspergillosis (n = 55), superficial aspergillosis (n = 5), allergic bronchopulmonary aspergillosis (n = 1), and from those colonized with Aspergillus (n = 12) were included in the study. The enzymatic activities were tested on solid media supplemented with the corresponding substrates. Elastase activity was detected in 95.6, 82.6, and 0% of A. fumigatus, A. flavus, and A. niger isolates, respectively. Acid proteinase activity was detected only for A. fumigatus and in 20 of 45 isolates belonging to this species. Phospholipase activity was present in all strains of A. fumigatus and A. niger but in none of the isolates of A. flavus. No statistical correlation could be established between the existence of elastase or acid proteinase activity and development of invasive disease (p > 0.05). While high phospholipase production was found to be associated with development of invasive aspergillosis (p < 0.01), not all isolates that caused invasive diseases showed high phospholipase activities.