Diversity of nontuberculous mycobacterial infections in Kuwait. Diversity of nontuberculous mycobacterial infections in Kuwait: rapid identification and differentiation of Mycobacterium species by multiplex PCR, INNO-LiPA Mycobacteria v2 assay and PCR-sequencing of rDNA

Objective: Non-tuberculous mycobacteria (NTM) often cause disease that is clinically indistinguishable from tuberculosis. Specific identification is important since treatment varies according to Mycobacterium species causing infection. This study used multiplex PCR (mPCR) assay for rapid differentiation of mycobacterial growth indicator tube 960 system (MGIT) cultures as Mycobacterium tuberculosis (MTB) or NTM. Further identification was achieved by INNO LiPA Mycobacteria v2 assay (LiPA) and/or PCR-sequencing of 16S-23S internally transcribed spacer (ITS) region of rDNA. Materials and Methods: DNA was extracted from MGIT cultures (n=1033) and mPCR was performed to differentiate MTB from NTM. LiPA was performed and results were interpreted according to kit instructions. ITS region was amplified and sequenced by using panmycobacterial primers. Results: mPCR identified 979 isolates as MTB, 53 isolates as NTM and one isolate as mixed culture. LiPA and/or PCR-sequencing confirmed 112 of 979 selected isolates as MTB. Mixed culture contained M. tuberculosis and M. fortuitum. LiPA yielded 12 patterns and identified 10 species/species complexes among 47 NTM, M. kansasii + M. scrofulaceum in mixed culture and five isolates only at genus level. PCR-sequencing yielded more accurate identification with 15 isolates correctly identified at species/subspecies level. Conclusions: mPCR rapidly differentiated MTB from NTM. LiPA correctly identified Mycobacterium species in 44 of 52 NTM isolates and two mixed cultures. PCR-sequencing yielded more accurate identification for 15 NTM important for proper patient management. Rapid differentiation as MTB or NTM by mPCR followed by species-specific NTM identification by LiPA/PCR-sequencing is most suitable for proper management of mycobacterial infections in Kuwait.