Evaluation of the INNO-LiPA Rif. TB assay, a reverse hybridization assay for the simultaneous detection of Mycobacterium tuberculosis complex and its resistance to rifampin

Abstract

Mycobacterium tuberculosis resistance to rifampin results from nucleotide changes in the gene encoding the beta-subunit of the RNA polymerase (rpoB). We developed a reverse hybridization-based line probe assay (LiPA; the INNO-LiPA Rif. TB) carrying one oligonucleotide probe for the detection of M. tuberculosis complex strains and nine probes designed to detect nucleotide changes in the relevant part of rpoB. This assay was evaluated with 107 M. tuberculosis isolates with known rpoB sequences, 52 non-M. tuberculosis complex strains, and 61 and 203 clinical isolates found to be sensitive and resistant, respectively, by in vitro testing. The results indicated that (i) the M. tuberculosis complex probe was 100% specific, (ii) when compared to the results of nucleotide sequencing, no discrepancies with the results of INNO-LiPA Rif. TB were observed, (iii) all strains sensitive by in vitro susceptibility testing were correctly identified, and (iv) among the strains resistant by in vitro susceptibility testing, only 4 (2%) yielded conflicting results. The INNO-LiPA Rif. TB is therefore a reliable and widely applicable assay and a valuable tool for routine diagnostic use, given its simplicity and rapid performance.

The occurrence of rare mutations in rifampicin-resistant clinical isolates from Kuwait

“…Most studies have reported nearly 5% of all phenotypically documented RIF-resistant strains with no detectable mutation in the N-terminal region and/or the RRDR of the rpoB gene [9][10][11][18][19][20][28][29][30][31][32]. The mutation responsible for RIF resistance in these strains could involve changes in other regions of the rpoB gene [18] or other genes whose products participate in antibiotic permeation or metabolism [7,8].…”

“…The molecular basis of resistance to RIF in nearly 95% of RIF-resistant strains is due to missense mutations or, less commonly, to small in-frame deletions or insertions in or around the 81 bp rifampicin resistance-determining region (RRDR) (corresponding to codons 507-533, Escherichia coli numbering system) of the rpoB gene, encoding the ␤-subunit of DNAdependent RNA polymerase [7,8]. Thus, several molecular methods have been developed for the rapid (1-2 days) detection of rpoB mutations in this region [8][9][10][11][12][13][14][15][16][17] and one of these, the line probe (INNO-LiPA Rif. TB) assay (LiPA) is available commercially [10,11].…”

“…Thus, several molecular methods have been developed for the rapid (1-2 days) detection of rpoB mutations in this region [8][9][10][11][12][13][14][15][16][17] and one of these, the line probe (INNO-LiPA Rif. TB) assay (LiPA) is available commercially [10,11]. Resistance in nearly 5% of the RIFresistant isolates either involves mutations in other regions of the rpoB gene such as codon V146 or is due to unknown mechanisms [7,18,19].…”

Comparison of rifabutin susceptibility and rpoB mutations in multi-drug-resistant Mycobacterium tuberculosis strains by DNA sequencing and the line probe assay

“…Numerous previous studies have shown that the majority of rifampicin-resistant isolates of M. tuberculosis are also isoniazid resistant [1]. The detection of rifampicin resistance therefore has the potential benefit of simultaneously detecting MDR-TB [1,2]. One of the commercial kits used to determine drug resistance is the INNO-LiPA Rif.…”

“…This assay is an excellent tool for detecting mutations in hot-spot regions of rpoB, a gene that encodes a subunit of RNA polymerase. Such mutations occur in up to 95% of rifampicin-resistant strains [2].…”