Population genomics confirms acquisition of drug-resistant Aspergillus fumigatus infection by humans from the environment.

Infections caused by the fungal pathogen Aspergillus fumigatus are increasingly resistant to first-line azole antifungal drugs. However, despite its clinical importance, little is known about how susceptible patients acquire infection from drug-resistant genotypes in the environment. Here, we present a population genomic analysis of 218 A. fumigatus isolates from across the UK and Ireland (comprising 153 clinical isolates from 143 patients and 65 environmental isolates). First, phylogenomic analysis shows strong genetic structuring into two clades (A and B) with little interclade recombination and the majority of environmental azole resistance found within clade A. Second, we show occurrences where azole-resistant isolates of near-identical genotypes were obtained from both environmental and clinical sources, indicating with high confidence the infection of patients with resistant isolates transmitted from the environment. Third, genome-wide scans identified selective sweeps across multiple regions indicating a polygenic basis to the trait in some genetic backgrounds. These signatures of positive selection are seen for loci containing the canonical genes encoding fungicide resistance in the ergosterol biosynthetic pathway, while other regions under selection have no defined function. Lastly, pan-genome analysis identified genes linked to azole resistance and previously unknown resistance mechanisms. Understanding the environmental drivers and genetic basis of evolving fungal drug resistance needs urgent attention, especially in light of increasing numbers of patients with severe viral respiratory tract infections who are susceptible to opportunistic fungal superinfections.

Molecular characterization of a subgroup IE intron with wide distribution in the large subunit rRNA genes of dermatophyte fungi.

Group I introns have the ability to catalyse their own excision (self-splice) from pre-RNA, and are found in a wide range of eukaryotic organisms. In fungal nuclear genomes, they have been identified in the small subunit (SSU) and large subunit (LSU) of the ribosomal RNA gene. Sequencing of the 3' region of the LSU rRNA gene of the dermatophyte Trichophyton interdigitale revealed a 393 bp group I intron, Tin.2563, containing the four characteristic conserved motifs (P,Q,R and S) essential for self-splicing. The predicted secondary structure revealed nine sets of conserved paired regions (P1-P9), with most similarity to a subgroup IE intron of the entomopathogenic hyphomycete Beauveria bassiana. Tin.2563 was inserted at a site in the LSU rDNA corresponding to position 2563 of the Escherichia coli 23S rRNA. PCR and sequence analysis showed an intron to be present at an identical location in the LSU rDNA of many dermatophytes, although its distribution was erratic. In contrast, an intron was present at the same location in multiple isolates (n = 20) of the clinically important anthrophilic species Trichophyton rubrum and T. interdigitale. Conservation of intron insertion site, subgroup and P helix sequences showed intron genotyping to be unsuitable for strain identification in dermatophytes. Phylogenetic analysis of intron sequences from different dermatophyte species indicated that lateral transfer of the element was likely to be a rare event.

High-frequency intragenomic heterogeneity of the ribosomal DNA intergenic spacer region in Trichophyton violaceum.

The intergenic spacer (IGS) of the rRNA genes was analyzed from the dermatophyte Trichophyton violaceum isolated from cases of tinea capitis in Taiwan and Iran. T. violaceum strains were cultured from different colonies, from single conidial colonies derived by dilution plating, and from micromanipulation of single conidia from clinical samples. A ribosomal DNA probe hybridizing to multiple EcoRI fragments was used to compare restriction fragment length polymorphisms in different T. violaceum isolates. The arthroconidia of T. violaceum that form in vivo during infection were shown to contain a single nucleus by 4',6'-diamidino-2-phenylindole staining. IGS regions from an isolate cultured from a single conidium were amplified, cloned, and sequenced. The results identified that heterogeneity exists between IGS regions within a single T. violaceum genome due to different copy numbers of a 171-bp tandem repeat. This suggests that the IGS of T. violaceum is partially excluded from the concerted evolution of the rRNA gene locus. The heterogeneous character of the IGS regions in T. violaceum contrasts with the closely related dermatophyte Trichophyton rubrum, posing further questions on the phylogeny and the evolution of dermatophyte fungi.

Serologic diagnosis of allergic bronchopulmonary aspergillosis in patients with cystic fibrosis through the detection of immunoglobulin G to Aspergillus fumigatus.

Allergic bronchopulmonary aspergillosis (ABPA) is seen in approximately 10% of patients with cystic fibrosis (CF) and can be difficult to diagnose. Consensus criteria require the presence of multiple elevated immunologic markers such as total immunoglobulin E (IgE), Aspergillus IgE and Aspergillus IgG, or precipitins for a robust diagnosis. There is some degree of standardization of total IgE and Aspergillus IgE levels, but there is no standardization in the measurement of IgG antibodies or precipitins to Aspergillus. The interpretation of results may, therefore, be confusing. Eighty-seven patients with CF were categorized as having ABPA or as controls, using the consensus criteria and an in-house enzyme immunoassay to measure IgG levels to Aspergillus. All sera from patients were then analyzed by commercial fluorescent immunoassay (FEIA) for the quantitative detection of anti-Aspergillus IgG. FEIA results were analyzed against the consensus conference minimum diagnostic criteria to ascertain a cutoff point, which could predict a diagnosis of ABPA in CF. Eighty patients with CF and with no or incomplete evidence of ABPA had a mean FEIA score of 51.1 mg/L, whereas 7 CF patients with ABPA had a mean FEIA score of 132.5 mg/L. Using receiver operator characteristic curve analysis of the ImmunoCAP (Phadia) IgG score on ABPA versus all other patients gave an area under the curve of 0.933 (estimated SE, 0.027). This analysis provisionally suggested that a score of 90 mg/L may be used as a cutoff point, which would give a sensitivity of 91% and specificity of 88.0% for the diagnosis of ABPA, though this requires further validation. This quantitative approach to Aspergillus IgG measurement in patients with CF along with the results of other tests will hopefully provide a more accurate approach to the diagnosis of ABPA.

Arthroconidia production in Trichophyton rubrum and a new ex vivo model of onychomycosis.

The dermatophyte fungus Trichophyton rubrum often produces arthroconidia in vivo, and these cells are thought to be involved in pathogenesis, and, in shed skin scales, to act as a source of infection. The purpose of this study was (i) to examine the environmental and iatrogenic factors which affect arthroconidiation in T. rubrum in vitro, (ii) to look at arthroconidia formation in a large number of clinical isolates of T. rubrum and (iii) to develop a new model for the study of arthroconidia formation in nail tissue. Arthroconidia production was studied in T. rubrum grown on a number of media and under conditions of varying pH, temperature and CO(2) concentration. The effect of the presence of antifungals and steroids on arthroconidia formation was also examined. Nail powder from the healthy toenails of volunteers was used as a substrate for arthroconidial production. On Sabouraud dextrose agar in the presence of 10 % CO(2) plus air, arthroconidial formation occurred optimally at 37 degrees C and pH 7.5, and was maximal at 10 days. Most isolates of T. rubrum showed a similar level of arthroconidial production, and only two out of 50 strains were unable to produce arthroconidia. Subinhibitory levels of some antifungals and betamethasone resulted in the stimulation of arthroconidia formation. Arthroconidial production in ground nail material also occurred under the same optimal conditions, but took longer to reach maximal levels (14 days). These in vitro and ex vivo results provide a useful basis for the understanding of arthroconidium formation in vivo in infected tissues such as nails.

PCR fingerprinting of Trichophyton mentagrophytes var. interdigitale using polymorphic subrepeat loci in the rDNA nontranscribed spacer.

The sequence of the nontranscribed spacer (NTS) region of the rDNA of Trichophyton mentagrophytes var. interdigitale strain 2111 was determined, and three individual subrepeat loci identified. The first repeat region contained eight tandem copies of a degenerate 33-43 bp sequence, whilst the second had two complete and two partial 300 bp repeats. The third locus contained six tandemly repetitive elements of between 67 and 89 bp, which showed sequence identity to the TrS2 repeats of Trichophyton rubrum. PCR amplification of the individual repetitive regions from 42 random isolates of T. mentagrophytes var. interdigitale identified fragment length polymorphisms at each locus. Sequence analysis of the PCR products revealed that the size variations resulted from differences in the copy number of each of the three sets of subrepeat elements, TmiS0, TmiS1 and TmiS2. In addition, some indels were present in the flanking regions of the TmiS1 repeats. Combining PCR fingerprints from each of the three polymorphic loci produced a total of 19 individual strain profiles. The method was rapid, reproducible and discriminatory, and the fragment patterns simple to interpret. PCR fingerprint analysis of variable tandem repeat loci in the T. mentagrophytes var. interdigitale NTS represents a valuable molecular typing method for future epidemiological investigations in this species.

Single strains of Trichophyton rubrum in cases of tinea pedis.

Multiple colonies of Trichophyton rubrum were isolated from single skin specimens from 10 patients with tinea pedis and were typed using a PCR-based analysis of repeats in the rRNA intergenic spacer. In each case only a single strain type of T. rubrum was isolated, suggesting a monotypic aetiology of tinea pedis. This is in contrast to the multiple strains previously shown to be involved in many cases of onychomycosis.

Retrospective survey of candidaemia in hospitalized patients and molecular investigation of a suspected outbreak.

Episodes of candida infection at a teaching hospital were investigated. During a 3-year period from 1998 to 2000, there were 53 cases of candidaemia. Candida albicans (64.2 %) was the most common causative species, followed by Candida glabrata (17.0 %) and Candida parapsilosis (15.1 %). Molecular analysis of a cluster of eight infections from a single unit was performed using Southern blotting with Ca3 probe hybridization. This showed that the patients were each infected by unrelated strains of C. albicans. On occasion, isolates were found to be closely related within individual patients. Following Southern blot analysis, it was concluded that the infections were not part of an outbreak caused by a single, epidemic strain.

British Society for Medical Mycology best practice recommendations for the diagnosis of serious fungal diseases.

Invasive fungal diseases are an important cause of morbidity and mortality in a wide range of patients, and early diagnosis and management are a challenge. We therefore did a review of the scientific literature to generate a series of key recommendations for the appropriate use of microbiological, histological, and radiological diagnostic methods for diagnosis of invasive fungal diseases. The recommendations emphasise the role of microscopy in rapid diagnosis and identification of clinically significant isolates to species level, and the need for susceptibility testing of all Aspergillus spp, if treatment is to be given. In this Review, we provide information to improve understanding of the importance of antigen detection for cryptococcal disease and invasive aspergillosis, the use of molecular (PCR) diagnostics for aspergillosis, and the crucial role of antibody detection for chronic and allergic aspergillosis. Furthermore, we consider the importance of histopathology reporting with a panel of special stains, and emphasise the need for urgent (<48 hours) and optimised imaging for patients with suspected invasive fungal infection. All 43 recommendations are auditable and should be used to ensure best diagnostic practice and improved outcomes for patients.

The use of Niger seed agar to screen for Candida dubliniensis in the clinical microbiology laboratory.

Candida dubliniensis is a recently described pathogenic yeast that is closely related to C. albicans. The germ tube test is used routinely in diagnostic laboratories for the identification of C. albicans, and C. dubliniensis may also produce germ tubes under the same conditions. We evaluated a previously described method for differentiating between the two species using Niger seed agar (Staib agar). The aim was to find a useful, user-friendly and cost-effective method for use in diagnostic work. C. albicans produces only yeast cells on this medium after 24 h at 37 degrees C, while C. dubliniensis produces extensive hyphal and pseudohyphal growth that is easily observed. Of 495 yeasts isolated in, or sent for identification to, a diagnostic mycology laboratory 9 isolates (1.8%) were found to be C. dubliniensis. The method was found to be valuable for screening yeasts before proceeding to further identification if positive for hyphal/pseudohyphal growth on Niger seed agar. This method is therefore suitable for the screening of selected yeast isolates in order to identity C. dubliniensis and will further our understanding of the clinical importance of this species.

Laboratory diagnosis of invasive aspergillosis: from diagnosis to prediction of outcome.

Invasive aspergillosis (IA), an infection caused by fungi in the genus Aspergillus, is seen in patients with immunological deficits, particularly acute leukaemia and stem cell transplantation, and has been associated with high rates of mortality in previous years. Diagnosing IA has long been problematic owing to the inability to culture the main causal agent A. fumigatus from blood. Microscopic examination and culture of respiratory tract specimens have lacked sensitivity, and biopsy tissue for histopathological examination is rarely obtainable. Thus, for many years there has been a great interest in nonculture-based techniques such as the detection of galactomannan, ß -D-glucan, and DNA by PCR-based methods. Recent meta-analyses suggest that these approaches have broadly similar performance parameters in terms of sensitivity and specificity to diagnose IA. Improvements have been made in our understanding of the limitations of antigen assays and the standardisation of PCR-based DNA detection. Thus, in more recent years, the debate has focussed on how these assays can be incorporated into diagnostic strategies to maximise improvements in outcome whilst limiting unnecessary use of antifungal therapy. Furthermore, there is a current interest in applying these tests to monitor the effectiveness of therapy after diagnosis and predict clinical outcomes. The search for improved markers for the early and sensitive diagnosis of IA continues to be a challenge.

Comparative genome analysis of Trichophyton rubrum and related dermatophytes reveals candidate genes involved in infection.

The major cause of athlete's foot is Trichophyton rubrum, a dermatophyte or fungal pathogen of human skin. To facilitate molecular analyses of the dermatophytes, we sequenced T. rubrum and four related species, Trichophyton tonsurans, Trichophyton equinum, Microsporum canis, and Microsporum gypseum. These species differ in host range, mating, and disease progression. The dermatophyte genomes are highly colinear yet contain gene family expansions not found in other human-associated fungi. Dermatophyte genomes are enriched for gene families containing the LysM domain, which binds chitin and potentially related carbohydrates. These LysM domains differ in sequence from those in other species in regions of the peptide that could affect substrate binding. The dermatophytes also encode novel sets of fungus-specific kinases with unknown specificity, including nonfunctional pseudokinases, which may inhibit phosphorylation by competing for kinase sites within substrates, acting as allosteric effectors, or acting as scaffolds for signaling. The dermatophytes are also enriched for a large number of enzymes that synthesize secondary metabolites, including dermatophyte-specific genes that could synthesize novel compounds. Finally, dermatophytes are enriched in several classes of proteases that are necessary for fungal growth and nutrient acquisition on keratinized tissues. Despite differences in mating ability, genes involved in mating and meiosis are conserved across species, suggesting the possibility of cryptic mating in species where it has not been previously detected. These genome analyses identify gene families that are important to our understanding of how dermatophytes cause chronic infections, how they interact with epithelial cells, and how they respond to the host immune response.

Restriction fragment length polymorphism analysis of ribosomal DNA intergenic regions is useful for differentiating strains of Trichophyton mentagrophytes.

Twenty isolates of Tricophyton mentagrophytes var. mentagrophytes and 47 isolates of T. mentagrophytes var. interdigitale, identified by morphological characteristics, were screened by restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified internal transcribed spacer (ITS) region of ribosomal DNA (rDNA). Sixty isolates (14 of 20 T. mentagrophytes var. mentagrophytes isolates and 46 of 47 T. mentagrophytes var. interdigitale isolates) shared an identical ITS RFLP profile and were further investigated by using a probe targeted to the rDNA nontranscribed spacer (NTS) region. Polymorphisms were observed in the NTS regions of both T. mentagrophytes var. mentagrophytes and T. mentagrophytes var. interdigitale isolates. Twenty-three individual RFLP patterns (DNA types P-1 to P-12 and A-1 to A-11) were recognized and divided into two groups depending on the presence (P) or absence (A) of a 2.5-kb band, which correlated to a large extent with the morphological variety. Eleven of 14 T. metagrophytes var. mentagrophytes isolates were A types, and all of the 46 T. mentagrophytes var. interdigitale isolates were P types. A majority of strains (23 of 60 [38.3%]) were characterized by one RFLP pattern (pattern P-1), and eight types (P-1 to P-6, P-8, and P-9) accounted for 75% (45 of 60) of all strains, including all of the T. mentagrophytes var. interdigitale isolates. The remaining 15 types were represented by one only isolate and included all of the T. mentagrophytes var. mentagrophytes isolates. We conclude that RFLP analysis of the rDNA NTS region is a valuable technique for differentiation of T. mentagrophytes strains. Furthermore, by use of this method, there appears to be a greater degree of diversity among T. mentagrophytes var. mentagrophytes isolates than among T. mentagrophytes var. interdigitale isolates.

Disruption of the gene encoding the ChiB1 chitinase of Aspergillus fumigatus and characterization of a recombinant gene product.

The gene encoding a major, inducible 45 kDa chitinase of Aspergillus fumigatus was cloned and analysis of the deduced amino acid sequence identified a chitinase of the fungal/bacterial class which was designated ChiB1. Recombinant ChiB1, expressed in Pichia pastoris, was shown to function by a retaining mechanism of action. That is, the beta-conformation of the chitin substrate linkage was preserved in the product in a manner typical of family 18 chitinases. Cleavage patterns with the N-acetylglucosamine (GlcNAc) oligosaccharide substrates GlcNAc(4), GlcNAc(5) and GlcNAc(6) indicated that the predominant reaction involved hydrolysis of GlcNAc(2) from the non-reducing end of each substrate. Products of transglycosylation were also identified in each incubation. Following disruption of chiB1 by gene replacement, growth and morphology of disruptants and of the wild-type strain were essentially identical. However, during the autolytic phase of batch cultures the level of chitinase activity in culture filtrate from a disruptant was much lower than the activity from the wild-type. The search for chitinases with morphogenetic roles in filamentous fungi should perhaps focus on chitinases of the fungal/plant class although such an investigation will be complicated by the identification of at least 11 putative active site domains for family 18 chitinases in the A. fumigatus TIGR database (http://www.tigr.org/).

Deep infection by Trichophyton rubrum in an immunocompromised patient.

Dermatophytes are common pathogens of skin but rarely cause invasive disease. We present a case of deep infection by Trichophyton rubrum in an immunocompromised patient. T. rubrum was identified by morphological characteristics and confirmed by PCR. Invasiveness was apparent by histopathology and immunohistochemistry. The patient was treated successfully with itraconazole.