Utility of fast mycobacterial interspersed repetitive unit-variable number tandem repeat genotyping in clinical mycobacteriological analysis.
BACKGROUND
Analysis of variable numbers of tandem repeats (VNTR) of genetic elements called mycobacterial interspersed repetitive units (MIRUs) is a recently described, polymerase chain reaction (PCR)-based method used to genotype Mycobacterium tuberculosis. It is much faster, requires a smaller amount of DNA, and has approximately the same discriminatory power as the standard IS6110 restriction fragment-length polymorphism (RFLP) method. We report the adaptation and optimization of MIRU-VNTR genotyping on a capillary electrophoresis system. We describe its application to 3 typical clinical situations encountered in our laboratory (Institut Pasteur de Bruxelles, Laboratoire Tuberculose et Mycobacteries; Brussels, Belgium).


METHODS
MIRU-VNTR genotyping was performed on heat-inactivated M. tuberculosis cultures obtained from clinical specimens on Lowenstein solid medium or in mycobacteria growth indicator liquid tubes (Becton Dickinson). After amplification of 12 genomic loci using 4 different multiplex PCRs, DNA fragments were separated by capillary electrophoresis using the ABI Prism 3100-Avant Genetic Analyzer (Applied Biosystems). Sizing of the PCR fragments and assignment of the various MIRU-VNTR alleles were done using the GeneScan and customized Genotyper software packages (PE Applied Biosystem).


RESULTS
Clustering on the basis of IS6110 fingerprinting of isolates from 3 different patients attending the same hospital was confirmed by MIRU-VNTR typing. This concordance between 2 independent, highly discriminatory techniques was decisive in triggering an epidemiological inquiry that led to identification of a bronchoscopy-related tuberculosis nosocomial infection. A mixed tuberculosis infection in a patient whose infection was initially suspected as a result of the IS6110 RFLP method was clearly identified by MIRU-VNTR typing. Finally, automated MIRU-VNTR analysis permitted the identification of laboratory contamination in 6 liquid cultures of M. tuberculosis within several hours.


CONCLUSIONS
These examples illustrate the utility of this genotyping technique for quick and accurate resolution of problems commonly encountered in clinical mycobacteriology.
Allix
Caroline
C
Institut Pasteur de Bruxelles, Laboratoire Tuberculose et Mycobacteries, Brussels, Belgium.
Supply
Philip
P
Fauville-Dufaux
Maryse
M
D016172:DNA Fingerprinting / Q000379:methods*; D004269:DNA, Bacterial / Q000235:genetics*; D019075:Electrophoresis, Capillary; D005838:Genotype; D006801:Humans; D018598:Minisatellite Repeats; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D014376:Tuberculosis / Q000382:microbiology
D004269:DNA, Bacterial