Diversity of Nontuberculous Mycobacteria In Kuwait: Rapid Identification And Differentiation Of Mycobacterium species by multiplex PCR, INNO-LiPA Mycobacteria v2 assay and PCR-sequencing of rDNA.

OBJECTIVE: Nontuberculous mycobacteria (NTM) often cause disease that is clinically indistinguishable from tuberculosis. Specific identification is important as treatment varies according to Mycobacterium species causing the 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 together with INNO LiPA Mycobacteria v2 assay (LiPA) and/or PCR-sequencing of rDNA for species-specific identification of selected MTB and all NTM isolates in Kuwait.

MATERIALS AND METHODS: DNA was extracted from MGIT cultures (n=1033) grown from 664 pulmonary and 369 extrapulmonary specimens from 1033 suspected tuberculosis patients. mPCR was performed to differentiate MTB from NTM. LiPA was performed and results were interpreted according to kit instructions. rDNA was amplified and sequenced by using panmycobacterial primers.

RESULTS: mPCR identified 979 isolates as MTB, 53 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 one culture and five isolates only at genus level. PCR-sequencing yielded more specific identification for 22 isolates at species/subspecies level.

CONCLUSIONS: mPCR rapidly differentiated MTB from NTM. LiPA identified 44 of 52 NTM isolates at species/species complex-level and two mixed cultures. PCR-sequencing yielded more specific identification at species/subspecies-level. Rapid differentiation as MTB or NTM by mPCR, followed by species-specific NTM identification by LiPA/PCR-sequencing is suitable for proper management of mycobacterial infections in Kuwait.