High-Throughput Mycobacterial Interspersed Repetitive-Unit–Variable-Number Tandem-Repeat Genotyping for Mycobacterium tuberculosis Epidemiological Studies

Abstract

dThe emergence of drug-resistant forms of tuberculosis (TB) represents a major public health concern. Understanding the transmission routes of the disease is a key factor for its control and for the implementation of efficient interventions. Mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) marker typing is a well-described method for lineage identification and transmission tracking. However, the conventional manual genotyping technique is cumbersome and time-consuming and entails many risks for errors, thus hindering its implementation and dissemination. We describe here a new approach using the QIAxcel system, an automated high-throughput capillary electrophoresis system that also carries out allele calling. This automated method was assessed on 1,824 amplicons from 82 TB isolates and tested with sets of markers of 15 or 24 loci. Overall allele-calling concordance between the methods from 140 to 1,317 bp was 98.9%. DNA concentrations and repeatability and reproducibility performances showed no biases in allele calling. Furthermore, turnaround time using this automated system was reduced by 81% compared to the conventional manual agarose gel method. In sum, this new automated method facilitates MIRU-VNTR genotyping and provides reliable results. Therefore, it is well suited for field genotyping. The implementation of this method will help to achieve accurate and cost-effective epidemiological studies, especially in countries with a high prevalence of TB, where the high number of strains complicates the surveillance of circulating lineages and requires efficient interventions to be carried out in an urgent manner.T uberculosis (TB) remains a major public health concern worldwide. The widespread emergence of multidrug resistant (MDR-TB) strains and extensively and extremely drug-resistant (XDR-TB and XXDR-TB) strains has hampered the management of treatment of the disease and the control of TB outbreaks (1). Many epidemiological questions remain unresolved, particularly in regard to the risk factors associated with the transmission of the bacilli to the host and to the prevalence of strain reactivation versus exogenous reinfection. Various genotype lineages of Mycobacterium tuberculosis have been described that can be associated with drug resistance, a higher rate of transmission, a higher rate of progression to disease, virulence, and vaccine escape (2, 3). The emergence of whole-genome sequencing (WGS) technologies can provide a complete genomic picture of lineage characterization and drug-resistant mutations. However, these technologies are presently not affordable for most laboratories, considering the costs and specialized skills required for data analysis. Thus, methods on the bench still have their value, as they have a faster turnaround time and can handle a higher batch capacity at a lower cost. One of these in particular, mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) marker typing, is commonly used for effective genotyping. There ...

Accuracy of the QIAxcel Automated System for MIRU-VNTR Genotyping of Mycobacterium tuberculosis in Two Limited Resource Settings

“…It would increase accuracy to 99.63% in Albania and 99.19% in Tunisia with a very high overall rate of concordance of 99.41%. These results are in line with other studies performed so far on MIRU-VNTR typing of MTBC with the QIAxcel system [14,15].…”

“…The QIAxcel system is an accurate method that detects allele lengths with a precision of 5 bp in more than half of the cases. For a proper allele calling, the maximum sizing deviation must not exceed half of the shortest repeat length [15]. Sizing deviation did not exceed 20 bp in Tunisia.…”

MIRU-VNTR Genotyping of Mycobacterium tuberculosis Strains Using QIAxcel Technology: A Multicentre Evaluation Study

“…tuberculosis isolates using 15- and 24 loci standard VNTR panels, overall concordance rate with the reference method was 98.9% demonstrating potential suitability of the system for routine M . tuberculosis genotyping [ 12 ].…”

Reproducibility of 15-Loci MIRU-VNTR Method in Mycobacterium tuberculosis Genotyping

“…The main objective of this study was to determine the reproducibility of 15-loci MIRU-VNTR method in different clinical samples isolated from confirmed patients. The DNA was extracted from the M. tuberculosis isolates by the boiling technique using 15 primers for MIRU-VNTR and PCR protocols has been used according to Gauthier et al (3). The PCR product was detected by the 1.5% agarose gel electrophoresis and the copy number of each repeated allele was determined in comparison with the H37RV PCR product as the standard strain.…”

“…Supply et al in the first paper published for the introduction of automated MIRU-VNTR method stated that 12-loci MIRU-VNTR typing was 100% reproducible, as all blinded duplicate samples analyzed were correctly assigned; they showed a small variation in the amplicon size of each locus in different repetitions (2). Gauthier et al confirmed the repeatability and reproducibility of the automated MIRU-VNTR method (3). None of these studies assessed the MIRU-VNTR reproducibility among different M. tuberculosis isolates taken from the same patient on different days or different clinical samples.…”