Background

Tuberculosis (TB) affirms its position as the preeminent cause of human death from infectious diseases, with an estimated 1.7 million deaths occurring globally each year, as reported by Daley [1]. The latest figures from the World Health Organization’s Global Tuberculosis Report 2023 reveal that 7.5 million new cases were diagnosed worldwide in 2022, surpassing pre-COVID levels. TB pathogen refers to a group of bacteria within the Mycobacterium tuberculosis complex (MTBC), including Mycobacterium tuberculosis (MTB), noted for its intricate drug resistance spectrum [2]. MTB, being the principal causative agent of TB, gains antibiotic resistance through gene mutations, significantly influencing clinical treatment approaches [3–5].

Surveillance of highly virulent and multidrug-resistant (MDR) MTB strains is imperative for securing precise diagnoses and potent treatment regimens [6]. Conventional culture-based phenotypic drug susceptibility testing (pDST) is the standard method for assessing MTB, although being time-consuming, typically requiring a span of days to weeks [3]. The prevalent molecular detection methods, including the real-time PCR-based Xpert MTB/RIF assay endorsed by the WHO, while being efficient, are limited to detecting known mutational hotspots and thus provide incomplete data on drug resistance [7]. Consequently, infections with MDR and extensively drug-resistant (XDR) MTB strains highlight an urgent need for advanced diagnostic tools to guide TB treatment options [8].

High-throughput sequencing technology has become a staple in clinical diagnostics. Nanopore sequencing, a cutting-edge method exemplified by the equipment from Oxford Nanopore Technologies (ONT), offers the benefits of long-read lengths, real-time data acquisition, and rapid sequencing [9]. While its accuracy has been a subject of improvement, significant strides in scientific and technological advancements have enhanced its performance in recent years [10,11]. Nanopore sequencing facilitates direct analysis of DNA or RNA sequences without the need for culturing MTB, enabling swift identification of MTB and detection of TB drug resistance. This approach might not only reduce the overall cost of MTB detection but also expedite the diagnostic process [12]. Therefore, the technology holds considerable promise for clinical applications and possesses a promising market outlook, making it an attractive option for clinical settings [13].

This study introduces a method that integrates multiplex PCR for targeted enrichment with nanopore sequencing, termed targeted nanopore sequencing. Furthermore, the bioinformatic tool TBProfiler is employed for drug resistance analysis, with its database encompassing 1,195 candidate mutations, which were obtained globally from more than 17,000 clinical isolates and assessed with whole-genome sequencing (WGS) and pDST [14].