Elisa Schena1, Lubov Nedialkova2, Emanuele Borroni1, Simone Battaglia1, Andrea Maurizio Cabibbe1, Stefan Niemann3, Christian Utpatel3, Matthias Merker3, Alberto Trovato1, Sabine Hofmann-Thiel4, Harald Hoffmann4, Daniela Maria Cirillo5. 1. Emerging Bacterial Pathogens Unit, Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan, Italy. 2. Institute of Microbiology and Laboratory Medicine, IML red GmbH, WHO Supranational Reference Laboratory of TB, Gauting, Germany. 3. Research Center Borstel, Molecular Mycobacteriology, German Center for Infection Research (DZIF), Partner Site Borstel, Borstel, Germany. 4. Institute of Microbiology and Laboratory Medicine, IML red GmbH, WHO Supranational Reference Laboratory of TB, Gauting, Germany Synlab MVZ Gauting, Gauting, Germany. 5. Emerging Bacterial Pathogens Unit, Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan, Italy cirillo.daniela@hsr.it.
Abstract
OBJECTIVES: The objective of this study was to develop standardized protocols for rapid delamanid drug susceptibility testing (DST) using the colorimetric resazurin microtitre assay (REMA) and semi-automated BACTEC™ MGIT™ 960 system (MGIT) by establishing breakpoints that accurately discriminate between susceptibility and resistance of Mycobacterium tuberculosis to delamanid. METHODS: MICs of delamanid were determined by the MGIT, the REMA and the solid agar method for 19 pre-characterized strains. The MIC distribution of delamanid was then established for a panel of clinical strains never exposed to the drug and characterized by different geographical origins and susceptibility patterns. WGS was used to investigate genetic polymorphisms in five genes (ddn, fgd1, fbiA, fbiB and fbiC) involved in intracellular delamanid activation. RESULTS: We demonstrated that the REMA and MGIT can both be used for the rapid and accurate determination of delamanid MIC, showing excellent concordance with the solid agar reference method, as well as high reproducibility and repeatability. We propose the tentative breakpoint of 0.125 mg/L for the REMA and MGIT, allowing reliable discrimination between M. tuberculosis susceptible and resistant to delamanid. Stop codon mutations in ddn (Trp-88 → STOP) and fbiA (Lys-250 → STOP) have only been observed in strains resistant to delamanid. CONCLUSIONS: We established protocols for DST of delamanid in the MGIT and REMA, confirming their feasibility in routine TB diagnostics, utilizing the same discriminative concentration for both methods. Moreover, taking advantage of WGS analysis, we identified polymorphisms potentially associated with resistance in two genes involved in delamanid activation.
OBJECTIVES: The objective of this study was to develop standardized protocols for rapid delamanid drug susceptibility testing (DST) using the colorimetric resazurin microtitre assay (REMA) and semi-automated BACTEC™ MGIT™ 960 system (MGIT) by establishing breakpoints that accurately discriminate between susceptibility and resistance of Mycobacterium tuberculosis to delamanid. METHODS: MICs of delamanid were determined by the MGIT, the REMA and the solid agar method for 19 pre-characterized strains. The MIC distribution of delamanid was then established for a panel of clinical strains never exposed to the drug and characterized by different geographical origins and susceptibility patterns. WGS was used to investigate genetic polymorphisms in five genes (ddn, fgd1, fbiA, fbiB and fbiC) involved in intracellular delamanid activation. RESULTS: We demonstrated that the REMA and MGIT can both be used for the rapid and accurate determination of delamanid MIC, showing excellent concordance with the solid agar reference method, as well as high reproducibility and repeatability. We propose the tentative breakpoint of 0.125 mg/L for the REMA and MGIT, allowing reliable discrimination between M. tuberculosis susceptible and resistant to delamanid. Stop codon mutations in ddn (Trp-88 → STOP) and fbiA (Lys-250 → STOP) have only been observed in strains resistant to delamanid. CONCLUSIONS: We established protocols for DST of delamanid in the MGIT and REMA, confirming their feasibility in routine TB diagnostics, utilizing the same discriminative concentration for both methods. Moreover, taking advantage of WGS analysis, we identified polymorphisms potentially associated with resistance in two genes involved in delamanid activation.
Authors: Suha Kadura; Nicholas King; Maria Nakhoul; Hongya Zhu; Grant Theron; Claudio U Köser; Maha Farhat Journal: J Antimicrob Chemother Date: 2020-08-01 Impact factor: 5.790
Authors: S Battaglia; A Spitaleri; A M Cabibbe; C J Meehan; C Utpatel; N Ismail; S Tahseen; A Skrahina; N Alikhanova; S M Mostofa Kamal; A Barbova; S Niemann; R Groenheit; A S Dean; M Zignol; L Rigouts; D M Cirillo Journal: J Clin Microbiol Date: 2020-10-21 Impact factor: 5.948
Authors: Alistair K Brown; Ahmed K B Aljohani; Fatimah M A Alsalem; Joseph L Broadhead; Jason H Gill; Yucheng Lu; Jonathan D Sellars Journal: Molecules Date: 2020-05-21 Impact factor: 4.411
Authors: Paola M V Rancoita; Federica Cugnata; Ana Luíza Gibertoni Cruz; Philip W Fowler; Daniela M Cirillo; Emanuele Borroni; Sarah J Hoosdally; Timothy M Walker; Clara Grazian; Timothy J Davies; Timothy E A Peto; Derrick W Crook Journal: Antimicrob Agents Chemother Date: 2018-08-27 Impact factor: 5.191