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.
Subject(s)
Bacterial Proteins , DNA, Ribosomal Spacer/genetics , Mycobacterium Infections/microbiology , Mycobacterium/classification , RNA, Ribosomal, 16S/genetics , RNA, Ribosomal, 23S/genetics , Reagent Kits, Diagnostic , Chaperonin 60 , Chaperonins/genetics , DNA Probes , Humans , Mycobacterium/genetics , Nucleic Acid Hybridization/methods , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length , Reference Standards , Reproducibility of Results , Sensitivity and Specificity , Species Specificity , Time FactorsABSTRACT
A panel of samples, previously typed by serology, was retyped using a line probe assay. One sample from a Brazilian Caucasian individual was serologically typed as B52/B39, but showed an aberrant HLA-B pattern on the diagnostic strip and was typed as B*52012/B*39new. Further analysis by allele-specific amplification and subsequent sequencing of exons 2 and 3 revealed a G(B*3908)-to-T nucleotide substitution at position 467 (codon 156) resulting in an Arg (B*3908)-to-Leu substitution. Furthermore, the sequence revealed a silent mutation at position 174 (codon 58): a G(B*3908)-to-A nucleotide switch. The sequence has been sent to the EMBL databank and the HLA Nomenclature Committee, and the allele was named B*3913.