Identification of non-tuberculous Mycobacteria clinical isolates to species level using four different advanced diagnostic technologies

Background: Infections by non- tuberculous mycobacteria (NTM) are of rising concern. Identification by phenotypic methods is time-consuming and challenging. In our work, we aimed to identify clinical NTM isolates to the species level using four different advanced diagnostic technologies. Methods: Clinical specimens underwent testing by Ziehl Neelsen (ZN) microscopy and GeneXpert®MTB/RIF assay. Suspected specimens for NTM were selected for inoculation on Lowenstein-Jensen medium (LJ) to obtain primary colonies. The isolated colonies were identified by matrix-assisted laser time-of-flight mass spectrometry (MALDI-TOF MS), and their DNA was extracted and tested by LCD-Microarray, 16SrRNA sequencing and Whole genome sequencing (WGS). Results: Out of total 112 specimens, 6 were presumptively identified as NTM (5.36%). WGS identified NTM isolates as 2 M. fortuitum, 2 M. abscessus, and 2 M. porcinum. The 16SrRNA sequencing agreed, except for 2 M. fortuitum isolates identified as the closely related M. neworleansense. MALDI-TOF MS identified M. fortuitum (n=3), and one of each of M. abscessus, M.porcinum, M. bacteremicum. The Microarray identified M. abscessus (n=2), Broad III group (n=3), however missed the identification of one isolate. An overall agreement of 66% was shown for 16SrRNA sequencing and MALDI-TOF, while 33% for LCD array with WGS in identification of NTM to the species level. Conclusion: The WGS remains the highest discriminatory identification method. The NTM isolates identified by the gold standard WGS were M. fortuitum, M. abscessus, and M. porcinum. The 16SrRNA sequencing and MALDI-TOF assay had the highest agreement with WGS in identification of NTM to species level.