Clinical application of target amplicon sequencing for rapid diagnosis of drug resistant mycobacterium tuberculosis in respiratory specimens

Abstract

Tuberculosis (TB) has been a global public health concern for more than a century. It remains one of the top 10 causes of death worldwide and the leading cause of death from a single infectious agent. Situation is worsened due to the emerging of multi-drug resistant TB (MDR-TB) and even extensively drug resistant TB (XDR-TB), dwarfing the effectiveness of current anti-TB treatment regime and the continuous effort towards TB infection control globally. Advancement in molecular diagnostics could provide a lot more rapid and accurate TB diagnosis and also drug-resistance detection comparing to the conventional methods including culture and phenotypic drug susceptibility test (pDST). This study aimed to evaluate a protocol for rapid TB diagnosis and drug resistance detection, with preliminary screening of MTB positive cases with IS6110 real-time PCR, followed by TB-drug resistance detection target amplicon sequencing NGS.

A total of 337 direct respiratory samples were collected during August 2020 to March 2021. All samples were tested with IS6110 real-time PCR and 76 samples tested with Ct values ranging 4.93 to 28.44 were selected for target amplicon sequencing, which covers 18 gene targets conferring anti-TB drug resistance. Target amplicon sequencing was performed using Illumina MiSeq on sample DNA purified and concentrated with our in-house validated protocol. Among the 76 selected samples, 69 of which were successfully sequenced and passed our defined acceptance criteria of 500X sequencing depth. Drug-resistance associated mutation(s) were detected in 17 samples with mutation frequency >95% and 1 sample with low mutation frequency at 37.04%, while no mutation was detected for the remaining 53 samples. Among the 17 samples detected with drug-resistance associated mutation(s) at frequency >95%, 10 of which were mono-drug resistant TB while the other 6 were multi-drug resistant TB. There were also 3 multi-drug resistant TB samples carrying mutations associated to fluoroquinolones (FQs) and considered as pre-XDR-TB. Concordant result was obtained in 16/17 (94.1%) when comparing the major variants at mutation frequency >95% with pDST results. One phenotypic pan-susceptible case was detected to harbour rpoB L533P mutation. The low variant frequency detected was phenotypic pan-susceptible. It could be well distinguished from the phenotypic resistant cases by variant frequency and hence the minor variants detected would not affect the overall result interpretation for target amplicon sequencing towards TB drug-resistance detection.

This evaluated protocol, with preliminary screening for IS6110 positive cases, followed by subsequent target amplicon sequencing for detection of drug-resistance associated mutations, could provide a rapid, accurate and cost-effective diagnostic solution for drug-resistance TB cases with turn-around time of around 10 working days. Which shortened the turn-around time a lot when comparing to the conventional methods including culture and phenotypic drug susceptibility test (pDST) which require 8 to 12 weeks. This protocol is also user friendly and easy to be adopted as routine testing by clinical laboratory worldwide. The timely genotypic resistance profile obtained from the target amplicon sequencing could facilitate clinician to have better and timely treatment planning for drug resistant TB patients and improve the epidemiology control of drug resistant TB.