A negative PCR result can be used to rule out IA and to limit the need for empirical antifungal therapy; thus, it has a role in diagnosing IA infections, especially in combination with antigen testing. PCR-positive cases classified as "false positives" regularly reflect the limitations of classic microbiological procedures or restricted use of consensus clinical methods employed to classify infection.

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.

Snake fungal disease (SFD) is an emerging disease of conservation concern in eastern North America. Ophidiomyces ophiodiicola, the causative agent of SFD, has been isolated from over 30 species of wild snakes from six families in North America. Whilst O. ophiodiicola has been isolated from captive snakes outside North America, the pathogen has not been reported from wild snakes elsewhere. We screened 33 carcasses and 303 moulted skins from wild snakes collected from 2010-2016 in Great Britain and the Czech Republic for the presence of macroscopic skin lesions and O. ophiodiicola. The fungus was detected using real-time PCR in 26 (8.6%) specimens across the period of collection. Follow up culture and histopathologic analyses confirmed that both O. ophiodiicola and SFD occur in wild European snakes. Although skin lesions were mild in most cases, in some snakes they were severe and were considered likely to have contributed to mortality. Culture characterisations demonstrated that European isolates grew more slowly than those from the United States, and phylogenetic analyses indicated that isolates from European wild snakes reside in a clade distinct from the North American isolates examined. These genetic and phenotypic differences indicate that the European isolates represent novel strains of O. ophiodiicola. Further work is required to understand the individual and population level impact of this pathogen in Europe.

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.

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.