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NGS for the identification of less frequently encountered mycobacteria species in a clinical laboratory

B KK Lo(1) A KS Tay(1) L H Sng(1,2)

1:Singapore General Hospital; 2:Duke-National University of Singapore

Nontuberculous mycobacteria (NTM) are increasingly becoming a major cause of pulmonary and extrapulmonary infections.  CTBL is a reference laboratory for identification of mycobacteria and unidentifiable species are frequently encountered in clinical samples. We assessed an in-house NGS workflow as a single assay for speciating mycobacteria unidentified by routine diagnostic methods. Clinical isolates (171 NTM, 5 M. tuberculosis), 1 NTM from heater-cooler water and 19 reference strains were sequenced using Illumina MiSeq. Majority of the NTM were unidentifiable or poorly identified by Vitek MS-RUO and line-probe assays; or had conflicting results. A CLC Genomics workflow program was used map the paired end reads to 4 in-house custom reference sequences made by concatenating 1) housekeeping genes: 16SrRNA-23SrRNA-rpoB and hsp65, 2) ITS, 3) M. avium complex IS and DT1 sequences and 4) erm41 sequences for M. abscessus complex; and identified via in-house BLAST using reference sequences. Definitive identification was assigned if at least 2 genes met the match criteria (a combination of the identification score and difference from the 2nd match). Isolates that remained unidentifiable or with ambiguous results were further analysed by BLAST searches of the mapped reads on NCBI. The agreement with isolate morphology for conventional tests was 64.8 versus 80.1% for NGS, and 91.3% for NGS with additional NCBI BLAST. The raw concordance between NGS and conventional tests was 55.1%, which improved to 69.4% after including manual correlation with phenotypic tests and online BLAST. NGS is potentially a suitable single assay for the identification of mycobacteria in the clinical laboratory.

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