Abstract
Tuberculosis (TB) remains a major global health challenge, particularly in resource-limited settings such as Fiji, where diagnostic capacity for drug-resistant Mycobacterium tuberculosis (M. tuberculosis) is constrained. This study evaluated a portable, targeted next-generation sequencing (tNGS) workflow using Oxford Nanopore’s MinION device, with the aim of enabling rapid detection of rifampicin and isoniazid resistance. The study progressed through four phases: optimisation of saliva decontamination, multiplex PCR development, sensitivity assessment using an attenuated MTB strain, and preliminary application to stored MDR-TB isolates from Fiji.
Phase I established NALC-NaOH (2%) as the most effective saliva decontaminant, offering a practical balance between contaminant suppression and preservation of amplifiable MTB DNA. In Phase II, a multiplex PCR assay targeting rpoB and katG achieved reliable co-amplification at an annealing temperature of 58 °C, reducing assay time to approximately 2.5 hours. Phase III demonstrated a limit of detection as low as 1 CFU/mL in spiked saliva, with sequencing results showing full concordance with the H37Rv reference genome. These findings highlight the potential of saliva as a non-invasive diagnostic specimen and confirm that portable sequencing platforms can deliver sensitivity approaching or exceeding current commercial assays. In Phase IV, testing on 18 archived MDR-TB isolates from Fiji showed consistent amplification, although only rpoB was visible in gel images. Nanopore sequencing failed to capture reads for the resistance-associated genes, with BLASTn analysis instead identifying non-mycobacterial sequences, likely reflecting insufficient mycobacterial DNA in the stored isolates.
This study provides proof-of-concept for a saliva-based workflow integrating multiplex PCR and nanopore sequencing to detect key resistance markers in MTB. While the approach demonstrated excellent sensitivity and specificity under controlled laboratory conditions, the Fiji trial underscores the importance of testing on fresh clinical material and highlights the challenges of applying this method to low-DNA or degraded samples. Collectively, these findings establish a strong foundation for further optimisation, expansion of resistance targets, and eventual clinical validation of portable sequencing workflows for TB control in resource-limited settings.