Christopher J. Linton

Molecular identification of pathogenic fungi

Systemic fungal infections represent a major cause of morbidity and mortality in immunocompromised patients. The ever-increasing number of yeast species associated with human infections that are not covered by conventional identification kits, and the fact that moulds isolated from deep infections are frequently impossible to identify using classical methods due to lack of sporulation, has driven the need for rapid, robust molecular identification techniques. We recently developed a rapid method of preparing fungal genomic DNAs using Whatman FTA filters, which has greatly facilitated molecular identification. Mould isolates cultured from dark grain mycetomas (destructive infections of skin/subcutaneous tissues that progress to involve muscle and bone) invariably fail to produce features by which they can be identified and were taxonomic mysteries. PCR amplification and sequencing of 250 bp of the internal transcribed spacer region 1 (ITS1) allowed us to distinguish between the known agents of mycetoma, to describe three new species associated with this disease and to define phylogenetic relationships. For yeasts, 153 isolates encompassing 47 species that had failed to be identified using classical methods were unambiguously identified by conventional sequencing of 350 bp of the 26S rRNA D1D2 region. These represented 5% of the isolates examined and included common species with atypical biochemical and phenotypic profiles, and rarer species infrequently associated with infection. Our recent studies indicate that FTA extraction coupled with pyrosequencing of 25 bp of ITS2 could potentially identify most common yeast species from pure culture in half a day. Together, these data underscore the importance of molecular techniques for fungal identification.

Candida nivariensis, an Emerging Pathogenic Fungus with Multidrug Resistance to Antifungal Agents

ABSTRACT In 2005, Candida nivariensis, a yeast species genetically related to Candida glabrata, was described following its isolation from three patients in a single Spanish hospital. Between 2005 and 2006, 16 fungal isolates with phenotypic similarities to C. nivariensis were submitted to the United Kingdom Mycology Reference Laboratory for identification. The strains originated from various clinical specimens, including deep, usually sterile sites, from patients at 12 different hospitals in the United Kingdom. PCR amplification and sequencing of the D1D2 and internal transcribed spacer 1 (ITS1) regions of the nuclear ribosomal gene cassette confirmed that these isolates from the United Kingdom are genetically identical to C. nivariensis. Biochemically, C. glabrata and C. nivariensis are distinguished by their differential abilities to assimilate trehalose. However, in contrast to the original published findings, we found that C. glabrata isolates, but not C. nivariensis isolates, are capable of assimilating this substrate. Antifungal susceptibility tests revealed that C. nivariensis isolates are less susceptible than C. glabrata isolates to itraconazole, fluconazole, and voriconazole and to have significantly higher flucytosine MICs than C. glabrata strains. Finally, C. nivariensis could be rapidly distinguished from the other common pathogenic fungus species by pyrosequencing of the ITS2 region. In the light of these data, we believe that C. nivariensis should be regarded as a clinically important emerging pathogenic fungus.

Molecular Identification of Unusual Pathogenic Yeast Isolates by Large Ribosomal Subunit Gene Sequencing: 2 Years of Experience at the United Kingdom Mycology Reference Laboratory

ABSTRACT Rapid identification of yeast isolates from clinical samples is particularly important given their innately variable antifungal susceptibility profiles. We present here an analysis of the utility of PCR amplification and sequence analysis of the hypervariable D1/D2 region of the 26S rRNA gene for the identification of yeast species submitted to the United Kingdom Mycology Reference Laboratory over a 2-year period. A total of 3,033 clinical isolates were received from 2004 to 2006 encompassing 50 different yeast species. While more than 90% of the isolates, corresponding to the most common Candida species, could be identified by using the AUXACOLOR2 yeast identification kit, 153 isolates (5%), comprised of 47 species, could not be identified by using this system and were subjected to molecular identification via 26S rRNA gene sequencing. These isolates included some common species that exhibited atypical biochemical and phenotypic profiles and also many rarer yeast species that are infrequently encountered in the clinical setting. All 47 species requiring molecular identification were unambiguously identified on the basis of D1/D2 sequences, and the molecular identities correlated well with the observed biochemical profiles of the various organisms. Together, our data underscore the utility of molecular techniques as a reference adjunct to conventional methods of yeast identification. Further, we show that PCR amplification and sequencing of the D1/D2 region reliably identifies more than 45 species of clinically significant yeasts and can also potentially identify new pathogenic yeast species.

Ultra-rapid preparation of total genomic DNA from isolates of yeast and mould using Whatman FTA filter paper technology – a reusable DNA archiving system

Conventional methods for purifying PCR-grade fungal genomic DNA typically require cell disruption (either physical or enzymatic) coupled with laborious organic extraction and precipitation stages, or expensive column-based technologies. Here we present an easy and extremely rapid method of preparing yeast and mould genomic DNAs from living cultures using Whatman FTA filter matrix technology. Aqueous suspensions of yeast cells or hyphal fragments and conidia (in the case of moulds) are applied directly (or after freeze-thawing) to dry FTA filters. Inoculated filters are then subjected to brief microwave treatment, to dry the filters and inactivate the organisms. Filter punches are removed, washed rapidly, dried and placed directly into PCR reactions. We show that this procedure inactivated all of the 38 yeast and 75 mould species tested, and generated PCR-grade DNA preparations in around 15 minutes. A total of 218 out of 226 fungal isolates tested liberated amplifiable DNA after application to FTA filters. Detection limits with yeast cultures were approximately 10 colony-forming units per punch. Moreover, we demonstrate that filter punches can be recovered after PCR, washed and used in fresh PCR reactions without detectable cross-contamination. Whatman FTA technology thus represents a cheap, ultra-rapid method of fungal genomic DNA preparation, and also potentially represents a powerful fungal DNA archiving and storage system.

Pyrosequencing Analysis of 20 Nucleotides of Internal Transcribed Spacer 2 Discriminates Candida parapsilosis, Candida metapsilosis, and Candida orthopsilosis

ABSTRACT Two new cryptic sister species, Candida orthopsilosis and Candida metapsilosis, were recently identified by consistent DNA sequence differences among several genes within the genetically heterogeneous Candida parapsilosis complex. Here, we present data demonstrating that Pyrosequencing analysis of 20 nucleotides of internal transcribed spacer region 2 rapidly and robustly distinguishes between these three closely related Candida species.

Rapid Molecular Identification of Pathogenic Yeasts by Pyrosequencing Analysis of 35 Nucleotides of Internal Transcribed Spacer 2

ABSTRACT Rapid identification of yeast species isolates from clinical samples is particularly important given their innately variable antifungal susceptibility profiles. Here, we have evaluated the utility of pyrosequencing analysis of a portion of the internal transcribed spacer 2 region (ITS2) for identification of pathogenic yeasts. A total of 477 clinical isolates encompassing 43 different fungal species were subjected to pyrosequencing analysis in a strictly blinded study. The molecular identifications produced by pyrosequencing were compared with those obtained using conventional biochemical tests (AUXACOLOR2) and following PCR amplification and sequencing of the D1-D2 portion of the nuclear 28S large rRNA gene. More than 98% (469/477) of isolates encompassing 40 of the 43 fungal species tested were correctly identified by pyrosequencing of only 35 bp of ITS2. Moreover, BLAST searches of the public synchronized databases with the ITS2 pyrosequencing signature sequences revealed that there was only minimal sequence redundancy in the ITS2 under analysis. In all cases, the pyrosequencing signature sequences were unique to the yeast species (or species complex) under investigation. Finally, when pyrosequencing was combined with the Whatman FTA paper technology for the rapid extraction of fungal genomic DNA, molecular identification could be accomplished within 6 h from the time of starting from pure cultures.

An Improved Protocol for the Preparation of Total Genomic DNA from Isolates of Yeast and Mould Using Whatman FTA Filter Papers

Here, we present a significantly improved version of our previously published method for the extraction of fungal genomic DNA from pure cultures using Whatman FTA® filter paper matrix technology. This modified protocol is extremely rapid, significantly more cost effective than our original method, and importantly, substantially reduces the problem of potential cross-contamination between sequential filters when employing FTA technology.

Polycytella Hominis is a mutated form of Scedosporium Apiospermum

PCR amplification and sequencing of two separate regions of the nuclear ribosomal repeat region revealed that Polycytella hominis, a hyphomycete isolated from a human case of mycetoma, was genetically indistinguishable from Scedosporium apiospermum (the anamorph of Pseudallescheria boydii). These organisms also exhibited remarkably similar susceptibility profiles to common antifungal agents. P. hominis is thus likely to be a mutant of S. apiospermum showing abnormalities of sporulation, for which a possible mechanism is discussed. Polycytella hominis should thus be regarded as a synonym of Scedosporium apiospermum.

Adiaspiromycosis Due to Emmonsia crescens is Widespread in Native British Mammals

Adiaspiromycosis caused by Emmonsia crescens is primarily a respiratory disease affecting small mammals, especially members of the Families Rodentia, Carnivora and Mustelidae. Although isolated reports exist of adiaspiromycosis in free-living British wildlife, the extent of infection in wild animals in the UK, and the significance of any associated pathology are unclear. Here, we report the results of histopathological examination of lungs of free-living wild mammals from the south–west UK coupled with digestion of lung material in potassium hydroxide followed by centrifugation and microscopic examination for the presence of adiaspores. The combined results showed that almost one-third (27/94, 28.7%) of animals examined had evidence of infection with E. crescens. Attempts to culture E. crescens from infected lungs were unsuccessful. However, E. crescens could be confirmed as the causative agent by PCR amplification and sequencing of DNA from adiaspores micro-dissected from animal lungs. The prevalence of adiaspiromycosis was largely independent of animal species or precise geography. Adiaspore burdens in most animals were low, consistent with transient exposure to E. crescens. However, burdens in several animals suggested heavy or repeated exposures to E. crescens, and were considered sufficient to have significantly impaired respiratory function. Finally, since E. crescens is apparently widespread in UK mammals and the first UK human case of adiaspiromycosis was reported recently, we present data obtained using a previous isolate of E. crescens demonstrating that both the mycelial and adiaspore phases of the organism are susceptible to amphotericin B, voriconazole, itraconazole and caspofungin.

Arthroderma olidum, sp. nov. A new addition to the Trichophyton terrestre complex

In 1981, four fungal isolates from hair of the European badger (Meles meles) were examined by Dr Phyllis Stockdale at the Commonwealth Mycological Institute, Kew, and deposited in the UK National Collection of Pathogenic Fungi as an undescribed member of the Trichophyton terrestre complex. The present paper formalizes the complete description of a new ascomycete taxon, Arthroderma olidum following successful recent attempts to re-isolate the same fungus from the soil of Badger holes in South West England. Furthermore, using ribosomal RNA gene sequencing, we show that the asexual form of A. olidum is conspecific with the recently described Trichophyton eboreum1 isolated from a human skin specimen in Germany.