<i>Mycobacterium abscessus</i> subspecies identification using the Deeplex Myc-TB targeted NGS assay

Abstract

M ycobacterium abscessus infections pose a significant clinical challenge, necessitat ing the development and testing of improved diagnostics.The Deeplex Myc-TB assay (Genoscreen, Lille, France) is a targeted next-generation sequencing (NGS) assay for the identification of Mycobacterium species and subspecies from clinical specimens.The assay targets ~400 bp of the hsp65 gene for identification of Mycobacterium species, together with portions of 18 additional genes, including rpoB, to determine drug resistance in the Mycobacterium tuberculosis complex (1).Sequence information from all of the gene targets is combined in a multilocus approach to aid in the identification of closely related Mycobacterium species.The aim of this study was to evaluate the Myc-TB assay's ability to differentiate the three subspecies of M. abscessus using culture isolates, with results compared to a line probe assay and whole-genome sequencing (WGS).The clinical need to differentiate the three subspecies of M. abscessus results in part from differences in macrolide susceptibility, including inducible resistance to macrolides in most M. abscessus subspecies abscessus and M. abscessus subspecies bolletii isolates mediated by erm(41), while M. abscessus subspecies massiliense does not typically possess inducible macrolide resistance due to a truncation in erm(41) (2, 3).Response rates for macrolide-based antibiotic therapy are typically lower for M. abscessus subsp.abscessus and subsp.bolletii compared to subsp.massiliense, and therefore, the identification of the subspecies can be useful to guide therapy while awaiting the results of 14 days of extended incubation of phenotypic broth susceptibility testing.Microbiologic culture isolates of M. abscessus were identified using matrix-assis ted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (4).M. abscessus isolates were lysed by placing a 1 L inoculating loop full of a colony into a tube containing 50 L of 0.1 mm silica glass beads and 100 L of 2.4 mm Zirconia beads (Biospec Products, Bartlesville, OK) and 500 L of double sterilized water.Tubes were heated at 95C-100C for 10 min and lysed on a Disruptor Genie (Scientific Industries, Bohemia, NY) for 2 min.Ten microliters of sample were diluted into 1,000 L of molecular grade water, and 200 L was extracted on an MP96 LC Instrument (Roche Applied Sciences, Indianapolis, IN) using the LC Pathogen Universal 200 protocol with an elution volume of 100 L.Nine microliters of extracted genomic DNA were amplified using the Deeplex Myc-TB following the manufacturer's instructions.NGS was performed using 0.2 ng of amplicon for the Nextera XT library preparation kit (Illumina, San Diego, CA), the MiSeq Reagent Kit V2. 300-cycle kit (Illumina), and the MiSeq platform (Illumina).FASTQ files were uploaded to the Genoscreen cloud-based analysis software (app.deeplex.fr).Phenotypic microbroth dilution susceptibility testing was performed following the CLSI microbroth dilution method, with readings performed at 3 and 14 days (5).The GenoType NTM-DR VER 1.0 line probe assay (Hain Lifesciences GMbH, Nehren, Germany)

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