Fungal Whole-Genome Sequencing for Species Identification: From Test Development to Clinical Utilization

Abstract

Using next-generation sequencing (NGS), we developed and validated a whole-genome sequencing (WGS)-based clinical test for fungal species identification on clinical isolates. The identification is mainly based on the fungal ribosomal internal transcribed spacer (ITS) region as the primary marker, and additional marker and genomic analysis applied for species within the Mucorales family (using the 28S rRNA gene) and Aspergillus genus (using the beta-tubulin gene and k-mer tree-based phylogenetic clustering). The validation study involving 74 unique fungal isolates (22 yeasts, 51 molds, and 1 mushroom-forming fungus) showed high accuracy, with 100% (74/74) concordance on the genus-level identifications and 89.2% (66/74) concordance on the species level. The 8 discrepant results were due to either the limitation of conventional morphology-based methodology or taxonomic changes. After one year of implementation in our clinical laboratory, this fungal NGS test was utilized in 29 cases; the majority of them were transplant and cancer patients. We demonstrated the utility of this test by detailing five case studies, in which accurate fungal species identification led to correct diagnosis, treatment adjustment or was ruled out for hospital acquired infection. This study provides a model for validation and implementation of WGS for fungal identification in a complex health system that serves a large immunocompromised patient population.

Exploring fungal diversity in Vietnam: A citizen science initiative

“…Initial identification of fungal isolates was performed using macroscopic and microscopic observation, with reference to the textbook “Description of Medical Mycology” [ 11 ]. Preliminary morphology-based fungal identification was then validated with additional methods, such as a proteomic method (Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry [MALDI-TOF MS]) or molecular methods (Polymerase Chain Reaction [PCR] assay and DNA sequencing), enabling identification to species level [ [12] , [13] , [14] , [15] , [16] , [17] ].…”

Metagenomic Sequencing of Positive Blood Culture Fluid for Accurate Bacterial and Fungal Species Identification: A Pilot Study

“…A more detailed algorithm of the scheme was described previously [ 8 ]. For the identification of fungal organisms, raw sequences from each sample were processed using the same workflow except that the internal transcribed spacer (ITS) region (encompassing ITS1, 5.8S rDNA, and ITS2) and 28S rRNA gene were used as marker genes [ 10 ]. These markers are widely used for the species identification of fungal organisms.…”

Outbreaks of Fungal Infections in Hospitals: Epidemiology, Detection, and Management

“…Logically, the position of WGS in mycological surveillance and tracking of outbreaks should be similar, but its implementation into routine workflows has been hindered by a number of factors. These are: (i) the substantively larger genomes of fungi (>35 Mbp for mould pathogens), (ii) the presence of ploidy, (iii) the likelihood of genome duplication and recombination events, (iv) the absence of well-developed standardized bioinformatic pipelines, and (v) the lack of comparative genome databases of an adequate breadth and depth [96][97][98]. Further, characteristics that lend themselves to constituting a reliable "reference" genome for many fungal species are not well documented, and standard reference genomes are not available, as they are for bacteria and viruses.…”