Dual DNA Barcoding for the Molecular Identification of the Agents of Invasive Fungal Infections

Abstract

Invasive fungal infections, such as aspergillosis, candidiasis, and cryptococcosis, have significantly increased among immunocompromised people. To tackle these infections the first and most decisive step is the accurate identification of the causal pathogen. Routine identification of invasive fungal infections has progressed away from culture-dependent methods toward molecular techniques, including DNA barcoding, a highly efficient and widely used diagnostic technique. Fungal DNA barcoding previously relied on a single barcoding region, the internal transcribed spacer (ITS) region. However, this allowed only for 75% of all fungi to be correctly identified. As such, the translational elongation factor 1α ( TEF1α ) was recently introduced as the secondary barcode region to close the gap. Both loci together form the dual fungal DNA barcoding scheme. As a result, the ISHAM Barcoding Database has been expanded to include sequences for both barcoding regions to enable practical implementation of the dual barcoding scheme into clinical practice. The present study investigates the impact of the secondary barcode on the identification of clinically important fungal taxa, that have been demonstrated to cause severe invasive disease. Analysis of the barcoding regions was performed using barcoding gap analysis based on the genetic distances generated with the Kimura 2-parameter model. The secondary barcode demonstrated an improvement in identification for all taxa that were unidentifiable with the primary barcode, and when combined with the primary barcode ensured accurate identification for all taxa analyzed, making DNA barcoding an important, efficient and reliable addition to the diagnostic toolset of invasive fungal infections.

Unambiguous identification of fungi: where do we stand and how accurate and precise is fungal DNA barcoding?

“…For plant- and animal/human-pathogenic or industrial fungi, a high level of taxonomic precision is required that cannot usually be achieved by phenotypic identifications. Instead, DNA barcoding or specific diagnostic testing and profiling have become indispensable (Criseo et al 2015 ; Crous et al 2015 , 2016 ; Irinyi et al 2015 ; Heim et al 2018 ; Hoang et al 2019 ). The emerging multi-drug resistant yeast Candida auris is one example of a fungus misidentified by phenotypic tools (Chatterjee et al 2015 ; Lockhart et al 2017 ).…”

“…In several fungal groups, ITS can only provide an initial approximation within a given clade, usually to a species complex, but cannot discriminate to the level of species. Two-marker barcoding systems, such as nuLSU/ITS and TEF1 for yeasts or human/animal pathogens, are a practicable solution in such cases (Kurtzman 2006 ; Robert et al 2011 ; Stielow et al 2015 ; Vu et al 2016 ; Hoang et al 2019 ), although the application of this approach in metabarcoding remains challenging.…”

DNA barcoding: a way forward to obtain deep insights about the realistic diversity of living organisms

“…However, the success rate of identifying the fungi was a bit low. Most recently this gap has been filled by introducing a secondary barcoding region, the translational elongation factor 1α (TEF1α) which ensured efficient and reliable detection of invasive fungal infections [57].…”

Exploring the Species Diversity of Edible Mushrooms in Yunnan, Southwestern China, by DNA Barcoding

“…To overcome the difficulties including the scarcity and ambiguity of morphological characteristics in identification, especially for closely related species, DNA sequences have become increasingly popular for species delimitation and identification [ 11 , 12 ]. Indeed, a number of diagnostic tools based on sequence variations have been developed for important groups of fungi [ 13 , 14 , 15 ].…”