The role of next generation sequencing in clinical microbiology diagnostics

Next Generation Sequencing (NGS) techniques are extensively used in research but application to clinical microbiology is still in its infancy. Because of the complexity of sequencing, some intrinsic to the technology and analysis, and some to the clinical interpretation of microbiology results, translation to clinical practice is challenging. This PhD-thesis examines the role of NGS in clinical microbiology diagnostics. Part one focusses on Whole Genome Sequencing (WGS) and describes how WGS was superior in differentiating outbreak from non-outbreak isolates compared to Amplified Fragment Length Polymorphism during a hospital outbreak with vancomycin resistant enterococci. Part two focusses on metagenomics for semi-quantitative pathogen detection from urine. A pilot study compared urine culture to urine metagenomics and investigated which metagenomic test characteristics were most comparable to culture outcomes. We developed an internal technical process control for clinical metagenomics in the form of DNA from the extremophile Thermus thermophilus. Next, we applied the hypotheses generated in the pilot study to a large prospective laboratory-based cohort of 529 urine samples and compared the metagenomics results to the results of semi-quantitative urine culture. We also explored antimicrobial drug resistance prediction from metagenomics and compared the results to phenotypic antimicrobial susceptibility testing. We also assessed the value of metagenomics in patients with a clinical syndrome in keeping with febrile UTI, in whom the diagnosis was only supported by a positive blood culture but in whom urine culture had remained negative. Finally, a summarising discussion and future directives are provided.