Evaluation of multiplex PCR coupled with DNA sequencing of 16S-23S internal transcribed spacer region for rapid differentiation and species-specific identification of non-tuberculous mycobacteria

Abstract number: P1816

Mokaddas E., Ahmad S.

Objective: Specific identification is of clinical relevance since treatment varies according to the mycobacterial species causing infection. The aim of this study was to evaluate a multiplex PCR (mPCR) assay for rapid differentiation of MGIT 960 culture isolates as non-tuberculous mycobacteria (NTM) or Mycobacterium tuberculosis complex (MTC) members and to confirm the NTM status of each isolate by DNA sequencing of 16S-23S internal transcribed spacer (ITS) region.

Methods: A total of 993 mycobacterial isolates grown with MGIT 960 system over a two-year period in Kuwait were used for evaluation. The DNA from heat-killed liquid cultures was extracted by incorporating removal of PCR inhibitors and mPCR targeting oxyR-ahpC intergenic region and rpoB gene was performed to differentiate NTM from MTC members. The 16S-23S ITS region was amplified and sequenced by using pan-mycobacterial primers. Mixed cultures were identified by a line probe assay.

Results: The mPCR identified 67 isolates (from 49 patients) as NTM, 924 isolates as MTC members and two isolates as mixed cultures. Thirteen different NTM species were identified. Of the 49 individual patient NTM isolates, 11, 10, 6, 5, 5, 2, 2 and 2 isolates were identified as M. fortuitum, M. kansasii, M. avium, M. intracellulare, M. abscessus, M. lentiflavum, M. gordonae, and M. chelonae, respectively. One isolate each was identified as M. chimaera, M. parascrofulaceum and M. immunogenum while two isolates could only be identified as Mycobacterium species. One NTM isolate contained a mixed culture, M. kansasii and M. scrofulaceum. The repeat NTM isolates recovered from some patients yielded identical results. The 150 randomly selected MTC isolates were identified as M. tuberculosis by 16S-23S ITS sequencing and/or hybridisation with M. tuberculosis-specific probes. The two mixed cultures identified by mPCR contained M. tuberculosis and M. fortuitum.

Conclusions: The mPCR accurately and rapidly differentiated all NTM isolates from MTC members. The DNA sequencing of 16S-23S ITS region led to species-specific identification of nearly all NTM isolates. Since majority of mycobacterial infections in developing countries are caused by MTC members, rapid differentiation of all mycobacterial isolates as NTM or MTC members by mPCR followed by species-specific identification of NTM by DNA sequencing is most suitable for proper management of mycobacterial infections.

Supported by KURA grant MI02/04.

Session Details

Date: 19/04/2008
Time: 00:00-00:00
Session name: 18th European Congress of Clinical Microbiology and Infectious Diseases
Location: Barcelona, Spain
Presentation type:
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