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

Minh Thuy Vi Hoang,Sharon C A Chen,Wieland Meyer,Laszlo Irinyi,Tania C Sorrell

doi:10.3389/fmicb.2019.01647

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.

Highlights

The PCR success rate was high within the 270 TEF1 sequences. 220 secondary barcodes were generated using the Al33F–Al33R primer pair and 50 were amplified with the Al34F–Al34R primer pair
The Al34F–Al34R primer set was required to amplify all strains of Aspergillus niger, Candida albicans, Candida dubliniensis, Kluyveromyes marxianus, and Pichia kudriavzevii
All sequences were submitted to the International Society for Human and Animal Mycology (ISHAM) Barcoding Database

Summary

Introduction

While AIDS-associated Pneumocystis jirovecii pneumonia (Pjp) and cryptococcosis have declined in developed countries due to widespread use of highly active antiretroviral treatment (Dromer et al, 2004; Morris et al, 2004; Rajasingham et al, 2017), the overall burden of invasive fungal diseases (IFDs), especially candidemia and invasive aspergillosis has increased worldwideDual DNA Barcoding of Mycoses (Patterson, 2005; Maschmeyer, 2006; Pfaller et al, 2006b; Warnock, 2007; Pappas et al, 2010; Benedict et al, 2017). The majority of the current fungal diagnostic techniques are inadequate for the identification of all pathogenic fungi, which is a pre-requisite for timely initiation of appropriate antifungal therapy (Schelenz et al, 2015; Irinyi et al, 2016; Cole et al, 2017). Culture-based identification techniques rely on morphological and phenotypic characteristics, are often inaccurate, and lack in most cases species-specific features. Morphology based diagnostics are time-consuming (7–14 days), highly laborious, and heavily dependent on the level of mycological expertise of the microscopist, making them unsuitable for rapid and reliable diagnosis (Irinyi et al, 2016)

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Conclusion