Abstract
Tuberculosis (TB) is the leading cause of death from infectious diseases, presenting a major threat to global public health. TB epidemiology highlights health inequities globally and locally, disproportionately affecting developing countries and marginalised communities such as M?ori, Pasifika, and refugees in low-TB burden countries like Aotearoa. The financial and health burdens associated with TB treatment limit patient adherence, contributing to increasing drug resistance. Subsequently, the World Health Organisation (WHO) recently recommended a new shorter oral regimen of drugs to treat multi-drug resistant TB (MDR-TB) and endorsed targeted next-generation sequencing (tNGS) as a diagnostic for MDR-TB. Oxford Nanopore sequencing is the best tNGS option for MDR-TB diagnostics due to its portability and affordability; however, its diagnostic capability for resistance to the oral regimen is unexplored.
We conducted a proof-of-concept study to evaluate the diagnostic capability of Oxford Nanopore Technologies for resistance to the new oral regimen. This involved designing primers and optimisation of PCR (polymerase chain reaction) conditions to amplify the genes conferring resistance (atpE, Rv0678, pepQ, ddn, fbiA, rrl, rplC) using purified gDNA from clinical TB samples (n = 3) as the template. We then purified PCR products and sequenced them on Oxford Nanopore MinION platform. Sequencing output was mapped to the Mycobacterium tuberculosis H37Rv reference genome using Geneious Prime to visualise and confirm accurate amplification. Gold standard Illumina sequencing platform was also used to validate accuracy.
Our results showed MinION can accurately and efficiently sequence genes conferring resistance to the oral regimen, allowing diagnosis of resistance status faster and cheaper than established tNGS platforms such as Illumina, which show equivalent accuracy under the same conditions. Combined with its decentralised design, this highlights MinION’s capacity to serve as an accessible diagnostic for MDR-TB, which is vital to combating TB in regions with limited infrastructure.