P05
Prospective evaluation of targeted next-generation sequencing of Mycobacterium tuberculosis complex strains in routine diagnostics in Germany.
D Nadarajan(1) P Beckert(2) M Kuhns(1) C Utpatel(2) I Barilar(2) V Dreyer(2,3) S Niemann(2,3) F P Maurer(1,3,4)
1:National and Supranational Reference Laboratory for Mycobacteria, Research Center Borstel, Leibniz Lung Center, Borstel, Germany; 2:Molecular and Experimental Mycobacteriology, Research Center Borstel, Borstel, Germany; 3:German Center for Infection Research (DZIF), Partner Site Hamburg-Lübeck-Borstel-Riems, Borstel, Germany; 4:Institute of Medical Microbiology, Virology and Hygiene, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
Tuberculosis (TB) challenges the global health care systems. To provide effective treatment regimens, rapid drug resistance prediction is crucial. This study prospectively evaluates targeted next-generation sequencing (tNGS) from primary patient material. Six DNA extraction and purification methods (BRUKER GenoLyse, BRUKER FluoroLyse, BD MAX MDR-TB, QIAGEN QIAamp DNA Micro Kit, Molbio Trueprep AUTO and a published protocol (George et al. 2020) for heat stabilization of mycobacterial DNA) from primary patient material were evaluated. Extracted DNAs were prescreened for TB, positive samples were subjected to tNGS (Deeplex Myc-TB, GenoScreen) and sequenced. While all DNA extraction methods led to acceptable tNGS success rate, differences in the quality and quantity of DNA obtained was observed. Using the Molbio Trueprep AUTO and MTB Plus PCR assay, we prospectively evaluated 977 (274 sputum and 703 non-sputum) patient samples between May 2021 and April 2022. In total, 88 (42 sputum and 46 non-sputum; smear positive n=32, smear negative n=56) TB positive samples were further analyzed with tNGS using the Trueprep AUTO DNA preparation. Full tNGS DST profiles were obtained for 28, insufficient for 5, failed for 40 samples. 15 samples were excluded prior to sequencing (no DNA fragment analyzer signal). tNGS from primary patient material can be included in a TB diagnostics routine work flow and provides comprehensive data on drug resistance prior to culture positivity. The comparably high cost per reaction, the need for expensive sequencing equipment and highly trained personnel has to be addressed to improve adoption of tNGS, especially in low resource countries.