Development and application of nanopore sequencing based methods for rapid, culture-free diagnosis of tuberculosis

Strinden, Michael (2022) Development and application of nanopore sequencing based methods for rapid, culture-free diagnosis of tuberculosis. Doctoral thesis, University of East Anglia.

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Tuberculosis (TB) is a condition of global health concern with an estimated 1/3 of the human population infected. A growing percentage of these infections also demonstrate resistance to antibiotics, increasing morbidity and mortality in affected populations. The gold standard for drug susceptibility testing (DST), microbial culture, is very slow (weeks-months) and can’t provide the necessary information within a clinically useful timeframe. Culture DST also requires specialist equipment that is not broadly available, therefore drug-resistant TB (DR-TB) is underdiagnosed globally. These limitations mean there is an urgent need for the development and uptake of new, rapid, DST technologies. Existing molecular technologies such as Xpert MTB/RIF offer rapid TB diagnosis but are only capable of detecting Rifampicin resistance due to limitations in PCR multiplexing technology. Comparatively, the GenoType MTBDRplus and MTBDRsl assays provide broader DST testing capability but are far from comprehensive for detecting all important drug-resistance associated mutations.

Targeted next-generation sequencing (tNGS) has the potential to rapidly diagnose TB and determine drug-resistance by amplification of known mutation loci. We developed a tNGS assay for DST covering 13 anti-tuberculous drugs using known SNPs (~200) in 16 Mycobacterium tuberculosis genes. Genotypic and phenotypic test performance were assessed during a blinded study of 392 contrived samples provided by the Foundation for Innovative New Diagnostics (FIND). This tNGS assay was found to have an overall genotypic sensitivity of 95% and specificity of 99% when compared to Illumina. The phenotypic sensitivity was 95%-97% and specificity was 96%-100% across all targeted drugs.

Clinical metagenomics has the potential to diagnose TB, perform DST, and provide epidemiological information directly from sputum in a single assay. We developed a metagenomic sequencing based TB test and evaluated it on spiked sputum samples from collaborators at the Norfolk and Norwich University Hospital (NNUH). Analysis showed commensal bacteria were present in high numbers, accounting for the majority of reads, thereby reducing analytical sensitivity. Attempts to design a commensal depletion protocol proved unsuccessful and metagenomic development was halted.

In conclusion, two approaches for rapid DST and TB diagnosis were designed and tested using contrived clinical samples. The tNGS method showed excellent potential for clinical use and is undergoing continued evaluation by FIND and the WHO under their Seq&Treat program. Continued development of the method has led to reductions in assay complexity, cost and turnaround time and use of the new WHO mutation list and simplified analysis tool will aid implementation of the test in the future.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Medicine and Health Sciences > Norwich Medical School
Depositing User: Chris White
Date Deposited: 16 Feb 2023 14:44
Last Modified: 16 Feb 2023 14:44


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