Rapid Identification of Mycobacteria to the Species Level Using INNO-LiPA Mycobacteria, a Reverse Hybridization Assay

Abstract

INNO-LiPA Mycobacteria (LiPA; Innogenetics, Zwijnaarde, Belgium) is a kit for the simultaneous detection and identification of Mycobacterium species in culture and identifies the Mycobacterium tuberculosis complex, the M. avium complex (MAC), and the following Mycobacterium species: M. kansasii, M. avium, M. intracellulare, M. scrofulaceum, M. gordonae, M. xenopi, and the M. chelonae-M. abscessus complex. The assay, which targets the 16S-23S rRNA spacer region, was evaluated on 157 mycobacterial strains that had been identified by conventional techniques and PCR-restriction enzyme analysis of the hsp65 gene (PRA). Forty-seven reference strains consisting of 37 different species and 110 human clinical isolates were submitted to the test, and all were hybridized with the Mycobacterium genus probe (MYC) on the LiPA strip (100% sensitivity). Ninety-four isolates hybridized to their corresponding species-or complex-specific probes; only one isolate phenotypically identified as M. gordonae did not react with its specific probe (99.4% accuracy). Thirty-seven MAC strains were phenotypically identified to the complex level and to the species level by LiPA as M. avium (n ‫؍‬ 18) or M. intracellulare (n ‫؍‬ 7) or as belonging to the M. avium-M. intracellulare-M. scrofulaceum complex (n ‫؍‬ 12). Of the last 12 strains, 10 had M. avium PRA patterns and 2 had M. intracellulare PRA patterns. Three isolates that had been identified as a single species by conventional identification were proven to be mixed cultures by the LiPA assay. The whole procedure can be performed in 1 working day, starting with the supernatant of a small amount of bacterial mass that had been treated by freezing and then boiling.To date, more than 70 mycobacterial species have been identified, and occasionally, isolates with unknown characteristics, mostly from immunodeficient patients, are being described. Differentiation of pathogenic and nonpathogenic mycobacteria other than Mycobacterium tuberculosis (MOTT) from members of the M. tuberculosis complex (MTC) is needed for patient management, considering that many MOTT are resistant to the antibiotics used for treatment of tuberculosis (27).Identification of mycobacterial isolates to the species level is performed by analysis of phenotypic and biochemical characteristics of the organisms after culture in solid media, which is a time-consuming process, or by high-pressure liquid chromatography analysis, which requires expensive equipment. Development of molecular tests has speeded up diagnosis, but most methods suffer from specific drawbacks. The AccuProbe system (Gen-Probe) differentiates only the MTC, M. avium, M. intracellulare, M. gordonae, and M. kansasii. In-house PCRbased identification systems have been developed but either identify a limited number of mycobacterial species or are difficult to use on a routine basis (4, 11). Restriction enzyme analysis of PCR products of specific genes is still widely used for identification of mycobacteria to the species level (7,19,23) and on clinical isolates (...

Nonconventional and new methods in the diagnosis of tuberculosis: feasibility and applicability in the field

“…It has been evaluated against DNA probes, conventional biochemical tests, and PCRrestriction fragment length polymorphism analysis showing clear-cut results [40,41] More recently the GenoType Mycobacterium assay (Hain Diagnostika, Nehren, Germany) for identification from clinical samples and liquid cultures has been introduced. It is based on amplification of the 16S-23S ribosomal DNA spacer region followed by hybridisation with 16 specific oligonucleotide probes; it has the advantage of detecting mixed mycobacterial infections [42].…”

Evaluation of GenoType and LiPA MYCOBACTERIA Assays for Identification of Finnish Mycobacterial Isolates

“…In previous reports, the LiPA assay has correctly identified more than 99.4% of isolates studied (12,21,25). The performance of the GenoType assay has not been evaluated before.…”

“…Currently, two DNA strip assays, INNOLiPA MYCOBACTERIA (Innogenetics N.V., Ghent, Belgium) (LiPA) The performance of the LiPA test has been assessed using BACTEC 12B bottles on a panel of clinical isolates from the United States (12) and on clinical isolates collected from Brazil (21) and Italy (25). To our knowledge, the GenoType test has not been evaluated previously.…”

A systematic review of rapid diagnostic tests for the detection of tuberculosis infection

“…122 Both Accuprobe and LIPA mycobacteria tests have been reported to have accuracies above 90%, but the cost of such probes limits their routine diagnostic use. 123,124 Nucleic acid amplication-based methods and DNA sequencing In developed countries, PCR-based assays are often routinely used to identify cultures such as the Hain DNA strip for the identification of M. TB complex and non-tuberculous mycobacteria (NTM) strains 125 or the INNO-LIPA assay for M. TB identification and rifampicin resistance. 122 Direct detection of M. TB in respiratory specimens by DNA sequencing is used to identify drug resistance.…”