Evaluation of a Commercial Line Probe Assay for Identification of Mycobacterium Species from Liquid and Solid Culture

Abstract

The performance of a commercial line probe assay (LiPA) (Inno-LiPA Mycobacteria; Innogenetics, Belgium) for the detection and identification of Mycobacterium species from liquid and solid culture was evaluated at five routine clinical laboratories. The LiPA method is based on the reverse hybridization principle, in which the mycobacterial 16S-23S ribosomal RNA (rRNA) spacer region is amplified by polymerase chain reaction (PCR). Amplicons are subsequently hybridized with oligonucleotide probes arranged on a membrane strip and detected by a colorimetric system. The test detects the presence of Mycobacterium species and specifically identifies Mycobacterium tuberculosis complex, Mycobacterium kansasii, Mycobacterium xenopi, Mycobacterium gordonae, Mycobacterium avium complex, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium scrofulaceum, and Mycobacterium chelonae - Mycobacterium abscessus complex. The results of LiPA were compared with the results obtained using traditional biochemical and molecular tests (DNA probe-based techniques, PCR restriction enzyme analysis of the 65 kDa heat-shock protein gene, and sequencing of the 16S rDNA). A total of 669 isolates, 642 of which were identified as Mycobacterium species and 27 as non- Mycobacterium species, were tested by LiPA. After analysis of 14 initially discordant results and exclusion of one isolate, concordant results were obtained for 636 of 641 Mycobacterium isolates (99.2% accuracy). All Mycobacterium species reacted with the MYC ( Mycobacterium species) probe (100% sensitivity), and all non- Mycobacterium species were identified as such (100% specificity).

Rapid Identification of Mycobacteria from Smear-Positive Sputum Samples by Nested PCR-Restriction Fragment Length Polymorphism Analysis

“…Recently, a paranitrobenzoic acid assay has been applied directly to clinical samples as a rapid screening assay for the detection of and differentiation between MTBC and NTM (32). Nevertheless, this approach does not allow further NTM species differentiation and has a long incubation time (3 weeks) before the results can be read.Recently, the development of PCR-based methods for the rapid identification and differentiation of mycobacterial organisms has significantly improved the diagnosis efficiency in terms of both sensitivity and specificity (3,5,12,14,17,22,24,26,27,30). We previously developed a multiplex nested PCR combined with lateral-flow technology for the rapid diagnosis of M. tuberculosis and MTBC isolates and the differentiation of these organisms from NTM organisms (28).…”

Comparative Evaluation of the New Version of the INNO-LiPA Mycobacteria and GenoType Mycobacterium Assays for Identification of Mycobacterium Species from MB/BacT Liquid Cultures Artificially Inoculated with Mycobacterial Strains

“…In previous reports, the first version of INNO-LiPA Mycobacteria was evaluated with success (8,10,11,18,(21)(22)(23) In our study, four M. intracellulare isolates were correctly identified by the INNO-LiPA and GenoType assays. For one M. intracellulare isolate that was identified as M. intracellulare sequevar Mac-A by the LiPA assay, the GenoType showed a noninterpretable hybridization pattern (positive hybridization results with bands 2 and 10).…”

“…Recently, new molecular biology techniques based on the PCR and reverse hybridization procedure have been marketed for mycobacterial identification (7,8,10,11,18,(21)(22)(23). The hybridization technique is performed on nitrocellulose strips onto which probe lines are fixed in parallel.…”

“…This format enables simultaneous detection and identification of different mycobacterial species. At present, two systems with this design are commercially available in Europe, the INNO-LiPA Mycobacteria test, designed to amplify the mycobacterial 16S-23S rRNA spacer region (8,10,11,18,(21)(22)(23), and the GenoType Mycobacteria assay, targeting the mycobacterial 23S rRNA (7). Any of these methods are available in the United States as Food and Drug Administration-approved tests and are used only for diagnosis.…”

“…This version of the test has been extensively evaluated with success for identification of mycobacteria isolated from solid and liquid cultures (8,10,11,18,(21)(22)(23).…”