P012
Detection of first-line and second-line Drug-Resistance mediating mutations in Mycobacterium tuberculosis with FluoroLyse and GenoXtract fleXT extracted DNA from sputum Using Next-Generation Sequencing
V Allerheiligen(1) L Wolf(1) M Eckart(1) R Spannaus(1) W Carbone(2)
1:Hain Lifescience GmbH - A Bruker company; 2:ELITechGroup S.p.A. - A Bruker company
Mycobacterium tuberculosis complex (MTBC) is a group of highly transmissible bacterial pathogens that cause significant worldwide morbidity and mortality, particularly among HIV-infected patients. Annually, MTBC is responsible for approximately 1.3 million deaths. The emergence of multi-drug-resistant (MDR) strains within the MTBC has significantly reduced treatment options. Current methods for detecting drug resistance in MTBC can be time-consuming, often requiring up to two months to yield results.
Next-generation sequencing (NGS) has provided valuable insights into the genetic diversity of MTBC, which are crucial for understanding the evolution and transmission of the disease. NGS has also facilitated the identification of drug-resistant strains, enabling rapid and accurate tailoring of treatment. Seven genes have been used for tuberculosis (TB) first-line and second-line drug resistance analysis: rpoB (Rifampicin), katG and inhA (Isoniazid), rrs (Amikacin), rrs and eis (Capreomycin), and gyrA and gyrB (Levofloxacin, Moxifloxacin).
NGS was performed using DNA from manual extraction (FluoroLyse, Hain Lifescience) or automated extraction (GXT fleXT, Hain Lifescience) from contrived NALC-NAOH decontaminated native sputum. Multiplex PCR targeting the seven resistance genes was employed for targeted NGS using Oxford Nanopore and Illumina technologies. Library preparation incorporated eight distinct barcodes on a single flow cell. Variant calling and annotation were performed using TB-Profiler. Concurrently, extracted DNA was analyzed with FluoroType MTBDR VER 2.0 and LiquidArray MTB-XDR VER 1.0 as references. Analytical sensitivity and sequencing quality were evaluated, demonstrating promising results.