Abstract
Shorter time-to-result is key for improving molecular-guided epidemiological investigation of tuberculosis (TB) cases. We performed a prospective study to evaluate the use of standardized MIRU-VNTR (mycobacterial interspersed repetitive-unit-variable-number tandem-repeat) typing of Mycobacterium tuberculosis directly on 79 fresh clinical samples from 26 TB patients consecutively enrolled over a 17-month period. Overall, complete 24-locus types were obtained for 18 out of the 26 (69.2%) patients and 14 of the 16 grade 3+ and grade 2+ samples (87.5%). The degree of completion of the genotypes obtained significantly correlated with smear microscopy grade both for 26 first samples (p = 0.0003) and for 53 follow-up samples (p = 0.002). For 20 of the 26 patients for whom complete or even incomplete M. tuberculosis isolate genotypes were obtained, typing applied to the clinical samples allowed the same unambiguous conclusions regarding case clustering or uniqueness as those that could have been drawn based on the corresponding cultured isolates. Standard 24 locus MIRU-VNTR typing of M. tuberculosis can be applied directly to fresh clinical samples, with typeability depending on the bacterial load in the sample.
Highlights
Molecular typing of pathogens has become an important tool in clinical microbiology and disease surveillance
PCR amplification of 24 MIRU-VNTR loci was performed with a standardized MIRU-VNTR Typing Kit, triplex version (GenoScreen, Lille, France), as described in the manufacturer’s manual
When full 24locus MIRU-VNTR results were not obtained in the first round, PCR and fragment analysis were repeated
Summary
Molecular typing of pathogens has become an important tool in clinical microbiology and disease surveillance. This is especially true for contagious diseases such as tuberculosis (TB). PCR amplification of 24 MIRU-VNTR loci was performed with a standardized MIRU-VNTR Typing Kit, triplex version (GenoScreen, Lille, France), as described in the manufacturer’s manual. ABI 3130 genetic analyzer (Applied Biosystems, USA), previously calibrated with the MIRU-VNTR Calibration Kit (GenoScreen, Lille, France). The reproducibility and accuracy of sizing were checked by analyzing the PCR fragments amplified from the M. tuberculosis H37Rv (or BCG, see above) positive control. When full 24locus MIRU-VNTR results were not obtained in the first round, PCR and fragment analysis were repeated. The MIRU-VNTR genotype result obtained from each clinical sample was compared with the MIRU-VNTR genotype obtained from the corresponding cultured M. tuberculosis isolate
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