Real-time Application of ITS and D1-D3 Nanopore Amplicon Metagenomic Sequencing in Fungal Infections: Enhancing Fungal Infection Diagnostics

While fungal infections cause considerable morbidity and mortality, the performance of the current diagnostic tests for fungal infection is low. Even though fungal metagenomics or targeted next-generation sequencing have been investigated for various clinical samples, the real-time clinical utility of these methods still needs to be elucidated. In this study, we used internal transcribed spacer (ITS) and D1-D3 ribosomal DNA nanopore amplicon metagenomic sequencing to assess its utility in patients with fungal infections. Eighty-four samples from seventy-three patients were included and categorized into 'Fungal infection,' 'Fungal colonization,' and 'Fungal contamination' groups based on the judgement of infectious disease specialists. In the 'Fungal infection' group, forty-seven initial samples were obtained from forty-seven patients. Three fungal cases detected not by the sequencing but by conventional fungal assays were excluded from the analysis. In the remaining cases, the conventional fungal assay-negative/sequencing-positive group (n=11) and conventional fungal assay-positive/sequencing-positive group (n=33) were compared. Non-Candida and non-Aspergillus fungi infections were more frequent in the conventional-negative/sequencing-positive group (p-value = 0.031). We demonstrated the presence of rare human pathogens, such as Trichosporon asahii and Phycomyces blakesleeanus. In the 'Fungal infection' group and 'Fungal colonization' group, sequencing was faster than culturing (mean difference = 4.92 days, p-value < 0.001/ mean difference = 4.67, p-value <0.001). Compared to the conventional diagnostic methods including culture, nanopore amplicon sequencing showed a shorter turnaround time and a higher detection rate for uncommon fungal pathogens.