| Author: | Lao, Hiu Yin |
| Title: | The clinical utility of nanopore sequencing for rapid diagnosis of acute invasive infection in normally sterile body sites |
| Advisors: | Siu, Gilman (HTI) |
| Degree: | Ph.D. |
| Year: | 2025 |
| Department: | Department of Health Technology and Informatics |
| Pages: | 199 pages : color illustrations |
| Language: | English |
| Abstract: | For a considerable period, culture has been the dominant method in clinical laboratories for pathogen identification. Nonetheless, the prolonged incubation period associated with culture significantly extends the sample-to-report time. In critical medical situations, like acute invasive infections, mortality rates escalate with delays. Empirical treatment prescriptions may result in suboptimal or ineffective treatment, while the utilization of broad-spectrum antimicrobials can promote selective pressure for antimicrobial-resistant strains. The advent of culture-independent nanopore sequencing, enabling long-read and real-time sequencing, presents a promising tool for rapid diagnosis in clinical laboratories. This study aimed to evaluate the clinical utility of nanopore sequencing for rapid diagnosis of acute invasive infections, with reference to traditional culture. Given the absence of a standardized protocol for interpreting sequencing data in clinical contexts, this study also aimed to establish a comprehensive nanopore-based sequencing workflow—from DNA extraction to data analysis. The detection threshold for distinguishing pathogens and background contaminants was determined. Furthermore, the clinical utility of nanopore targeted sequencing and unbiased metagenomic sequencing was evaluated and compared. The performance of nanopore 16S rRNA gene sequencing (Nanopore 16S) was first evaluated by comparing with the traditional Sanger (Sanger 16S) and short-read Illumina 16S rRNA (Illumina 16S) gene sequencing in taxonomic assignment of 172 MALDI-TOF MS-unidentifiable clinical isolates. It was found that the diagnostic accuracy of Nanopore 16S was 96.36%, identical to that of Sanger 16S, and much higher than that of Illumina 16S (69.07%). Despite the lower read accuracy, sequencing the full-length 16S rRNA gene using Nanopore 16S provided better taxonomic resolution compared to the short-read Illumina 16S, which only sequenced the V3-V4 region in the commercially available kit. Additionally, the study demonstrated the potential of sequencing technologies to uncover novel species, leading to the confirmation of a novel species, Scrofimicrobium appendicitidis, through whole-genome sequencing and phylogenomic analysis. Subsequently, the performance of Nanopore 16S for direct pathogen identification in normally sterile body fluids was evaluated with reference to culture results. Additionally, the performance of three analysis pipelines, including Epi2me, Emu, and NanoCLUST, was compared. Results showed that Nanopore 16S coupled with Emu demonstrated the highest concordance with the culture results. The concordance between culture and Emu was 97.7% among the 128 monomicrobial samples, compared to 85.2% for Epi2me and 79.7% for NanoCLUST. For the 230 cultured species in the 65 polymicrobial samples, Emu correctly identified 81.7% of cultured species, compared to 75.7% for Epi2me and 54.3% for NanoCLUST. To differentiate potential pathogens from background in Nanopore 16S, a threshold of relative abundance (TRA) at 0.058 was established through ROC analysis of the monomicrobial samples. However, a threshold could not be determined for the polymicrobial samples, it was presented as a random classifier in ROC analysis. The limit of detection of Nanopore 16S was found to be 90 CFU/ml. Nanopore targeted sequencing (NTS) and nanopore metagenomic sequencing (NMgS) workflows were developed and their performance for pathogen identification and AMR detection were compared with culture results. Of the 229 species cultured from 138 body fluids, NTS successfully identified 80.35% of the species, with 79.48% meeting the threshold of 0.058 TRA and 74.24% having a minimum of 10 classified reads. In contrast, NMgS identified 60.70% of the cultured species in the 138 body fluids, with only 41.48% of samples surpassing the threshold of 10 in Bracken. Among the 20 samples containing AMR ESKAPE pathogens, NTS detected associated AMR genes in 14 samples (70.0%). Out of the 24 AMR ESKAPE pathogens within these 20 samples, NMgS successfully identified AMR genes in association with 6 of the ESKAPE pathogens (25.0%). |
| Rights: | All rights reserved |
| Access: | open access |
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