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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) I Friesen(1) S Niemann(2,3) F P Maurer(1,4)

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

To provide effective tuberculosis (TB) treatment, rapid diagnostics and drug susceptibility testing (DST) is crucial. Targeted next-generation sequencing (tNGS) from primary patient material can bridge the gap between cultural DST and nucleic acid amplification tests. In this study, six commercial and published DNA extraction and purification methods (BRUKER GenoLyse and FluoroLyse, BD MAX MDR-TB, QIAGEN QIAamp DNA Micro Kit, Molbio Trueprep AUTO and a published protocol (George et al. 2020)) from primary patient material were evaluated. Extracted DNAs were prescreened for TB, positive samples were subjected to tNGS (Deeplex Myc-TB, GenoScreen) and sequenced. All tested DNA extraction methods led to acceptable tNGS success rate, differences in the quality and quantity of DNA obtained was observed. For prospective evaluation we analyzed 1231 (354 sputum and 877 non-sputum) patient samples between May 2021 and December 2022 using the Molbio Trueprep AUTO DNA extraction and MTB Plus PCR assay. In total, 90 (41 sputum and 49 non-sputum; 36 smear positive, 50 smear negative, 4 no data) TB positive samples were further analyzed with tNGS. Full tNGS DST profiles of Molbio MTB Plus positive samples were obtained for 29, insufficient for 6, failed for 37 and 18 samples were excluded prior to sequencing. tNGS from primary patient material can be included in routine TB diagnostics and provides comprehensive DST data prior to culture positivity. However, the implementation of tNGS, especially in low resource countries, is impaired by the comparably high cost per sample, the need for expensive sequencing equipment and highly trained personnel.

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