Direct Identification of Mycobacteria from MB/BacT Alert 3D Bottles: Comparative Evaluation of Two Commercial Probe Assays

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Abstract
The new INNO-LiPA Mycobacteria (Innogenetics, Ghent, Belgium), a reverse-hybridization-based line probe assay, and the AccuProbe assay (Gen-Probe Inc., San Diego, Calif.) were applied to MB/BacT Alert 3D (MB/BacT) system (Organon Teknika, Boxtel, The Netherlands) culture bottles and evaluated for mycobacterial identification. From 2,532 respiratory and extrapulmonary specimens submitted for culture, 168 were flagged positive by the MB/BacT system and promptly evaluated for identification (within 24 h). Each of 163 vials grew one mycobacterial isolate, including Mycobacterium tuberculosis complex ( n = 73), M. avium complex ( n = 3), M. avium ( n = 8), M. intracellulare ( n = 5), M. kansasii ( n = 15), M. gordonae ( n = 8), M. malmoense ( n = 3), M. chelonae ( n = 13), M. abscessus ( n = 2), M. xenopi ( n = 11), M. scrofulaceum ( n = 2), M. fortuitum ( n = 7), M. terrae ( n = 3), M. simiae ( n = 2), M. celatum ( n = 3), M. flavescens ( n = 1), M. interjectum ( n = 1), M. bohemicum ( n = 1), and M. pulveris ( n = 2). Five cultures yielded mixed growth of two mycobacterial species: M. tuberculosis complex plus M. gordonae ( n = 2), M. tuberculosis complex plus M. chelonae ( n = 1), M. tuberculosis complex plus M. xenopi ( n = 1), and M. avium plus M. chelonae ( n = 1). In testing of one-isolate vials, both systems showed excellent sensitivity and specificity for all species and complexes for which they are licensed (nine for INNO-LiPA Mycobacteria versus six for AccuProbe). There were minor discrepancies in results for two isolates identified by INNO-LiPA Mycobacteria as M. avium - M. intracellulare - M. scrofulaceum (MAIS) complex and by AccuProbe as M. intracellulare . In testing of two-isolate vials, INNO-LiPA Mycobacteria correctly identified all isolates, while the AccuProbe assay failed to identify three M. tuberculosis complex isolates and one M. avium isolate. The AccuProbe assay was completed within 2 h, while INNO-LiPA Mycobacteria required a 6-h period. In our opinion, INNO-LiPA Mycobacteria offers the following advantages: (i) it contains a genus-specific probe that, in addition to being used in genus identification, may be used as an internal control for both the amplification and hybridization steps; (ii) it simultaneously identifies M. tuberculosis complex, MAIS complex, and seven other mycobacterial species, even from mixed cultures; (iii) its mycobacterial DNA amplification ensures reliable results independent from the concentration of viable microorganisms; and (iv) it genotypically identifies M. kansasii and M. chelonae . In conclusion, even though INNO-LiPA Mycobacteria is considerably less easy to use than AccuProbe, requiring personnel skilled in molecular biology techniques, it represents an excellent approach for routine identification of frequently encountered mycobacteria.