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.
Conclusion: These examples illustrate the utility of this genotyping technique for quick and accurate resolution of problems commonly encountered in clinical mycobacteriology.