Evaluation of the LiPA MYCOBACTERIA Assay for Identification of Mycobacterial Species from BACTEC 12B Bottles

Abstract

The LiPA MYCOBACTERIA (Innogenetics NV, Ghent, Belgium) assay was used to identify mycobacterial isolates using culture fluid from positive BACTEC 12B bottles. The LiPA method involves reverse hybridization of a biotinylated mycobacterial PCR fragment, a 16 to 23S rRNA spacer region, to oligonucleotide probes arranged in lines on a membrane strip, with detection via biotin-streptavidin coupling by a colorimetric system. This system identifies Mycobacteriumspecies and differentiates M. tuberculosis complex,M. avium-M. intracellulare complex, and the following mycobacterial species: M. avium, M. intracellulare, M. kansasii, M. chelonaegroup, M. gordonae, M. xenopi, and M. scrofulaceum. The mycobacteria were identified in the laboratory by a series of tests, including the Roche AMPLICOR Mycobacterium tuberculosis (MTB) test, the Gen-Probe ACCUPROBE, and a PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the 65-kDa heat shock protein gene. The LiPA MYCOBACTERIA assay detected 60 mycobacterium isolates from 59 patients. There was complete agreement between LiPA and the laboratory identification tests for 26 M. tuberculosis complex, 9 M. avium, 3 M. intracellulare complex, 3 M. kansasii, 4 M. gordonae, and 5 M. chelonae group (all were M. abscessus) isolates. Three patient samples were LiPA positive forM. avium-M. intracellulare complex, and all were identified as M. intracellulare by the PCR-RFLP analysis. Seven additional mycobacterial species were LiPA positive forMycobacterium spp. (six were M. fortuitum, and one was M. szulgai). The LiPA MYCOBACTERIA assay was easy to perform, and the interpretation of the positive bands was clear-cut. Following PCR amplification and gel electrophoresis, the LiPA assay was completed within 3 h.

Proposal to elevate the genetic variant MAC-A, included in the Mycobacterium avium complex, to species rank as Mycobacterium chimaera sp. nov.

“…The aggregation, within the MAC, of strains that, although not identical, shared major traits arose from the impossibility of their differentiation by means of biochemical tests. While the commercialization of AccuProbe seemed to have resolved, at the level of routine diagnostic purposes, the dilemma of the distinction of M. avium from M. intracellulare and of both from MAIX, questions again arose with the subsequent introduction of LiPA, mainly because of the presence of strains with discrepant hybridization characteristics (Makinen et al, 2002;Suffys et al, 2001;Mijs et al, 2002b;Miller et al, 2000;Scarparo et al, 2001;Tortoli et al, 2001). In fact, strains do exist that are identified by AccuProbe as M. intracellulare while they are located by LiPA within the MAIS group, however, excluding their belonging to the species M. avium, M. intracellulare or M. scrofulaceum.…”

Detection of mixed infections with Mycobacterium lentiflavum and Mycobacterium avium by molecular genotyping methods

“…For genetic analysis, part of the sample was submitted to three consecutive cycles of snap freezing and boiling in 10 mM Tris/HCl, 1 mM EDTA and 1 % Triton X-100. (Miller et al, 2000); this assay was evaluated in Brazil with excellent results (Suffys et al, 2001). The PCR-restriction enzyme analysis (PRA) assay was originally described by Telenti et al (1993) and is based on amplification of part of hsp65 and restriction analysis with HaeIII and BstEII; evaluation of the assay in Brazil also demonstrated the straightforwardness of the method (da Silva Rocha et al, 2002).…”

Evaluation of the INNO‐LiPA mycobacteria v2 assay for identification of aquatic mycobacteria

“…This is the first study in which this molecular method has been used to identify aquatic mycobacteria isolated from fish. There are a number of reports in the literature which used both this and an earlier version of the LiPA kit to identify non-aquatic mycobacterial cultures obtained from limited geographical areas in the United States of America (Miller, Infante & Cleary 2000), Finland (Mäkinen, Sarkola, Marjamaki, Viljanen & Soini 2002) and Italy (Tortoli, Nanetti, Piersimoni, Cichero, Farina, Mucignat, Scarparo, Bartolini, Valentini, Nista, Gesu, Tosi, Crovatto & Brusarosco 2001;Tortoli, Mariottini & Mazzarelli 2003). Genetic heterogeneity of mycobacteria has been associated with differences in geographical origin (Alcaide, Richter, Bernasconi, Springer, Hagenau, Schulze-Robbecke, Tortoli, Martin, Bottger & Telenti 1997;Devallois, Picardeau, Paramasivan, Vincent & Rastogi 1997).…”