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

Abstract

BackgroundMolecular genotyping of M.tuberculosis is an important laboratory tool in the context of emerging drug resistant TB. The standard 24-loci MIRU-VNTR typing includes PCR amplification followed by the detection and sizing of PCR fragments using capillary electrophoresis on automated sequencers or using agarose gels. The QIAxcel Advanced system might offer a cost-effective medium-throughput alternative.MethodsPerformance characteristics of the QIAxcel Advanced platform for the standard 24 VNTR loci panel was evaluated at two centres on a total of 140 DNA specimens using automated capillary electrophoresis as a reference method. Additionally 4 hypervariable MIRU-VNTR loci were evaluated on 53 crude DNA extracts. The sizing accuracy, interlaboratory reproducibility and overall instrument’s performance were assessed during the study.ResultsAn overall concordance with the reference method was high reaching 98.5% and 97.6% for diluted genomic and crude DNA extracts respectively. 91.4% of all discrepancies were observed in fragments longer than 700bp. The concordance for hypervariable loci was lower except for locus 4120 (96.2%). The interlaboratory reproducibility agreement rates were 98.9% and 91.3% for standard and hypervariable loci, respectively. Overall performance of the QIAxcel platform for M.tuberculosis genotyping using a panel of standard loci is comparable to that of established methods for PCR fragments up to 700bp. Inaccuracies in sizing of longer fragments could be resolved through using in-house size markers or introduction of offset values. To conclude, the QiaXcel system could be considered an effective alternative to existing methods in smaller reference and regional laboratories offering good performance and shorter turnaround times.

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

“…These variations particularly affected loci 2163b and 4052. According to the authors of this study, the accuracy depended primarily on the PCR fragment length being suboptimal for sizes >750 bp [8]. These problems could be both loci-specific and PCR fragment size-dependent, as observed in our study.…”

“…The new approach to variable number tandem repeats (VNTR) analysis using the QIAxcel capillary electrophoresis system and a software-integrated peak calling function was first reported in Japan in 2013 [14]. The multicenter study conducted in two references centers in London and Milan [8] compared the QIAxcel genotyping results with the reference technique and demonstrated that PCR fragment sizes varied significantly depending on the number of copies within specific VNTR loci with the shortest and longest fragments. These variations particularly affected loci 2163b and 4052.…”

“…These problems could be both loci-specific and PCR fragment size-dependent, as observed in our study. As previously recommended, an optimization of separation procedures and peak calling algorithms, especially for the longest fragments, are necessary [8].…”

“…Regarding MIRU-VNTR typing of MTBC isolates, the QIAxcel system is proposed as an affordable tool that could replace conventional gel electrophoresis and provide high concordance with the reference methods. A fairly recent multicenter evaluation study carried out in two large reference centers in UK and Italy [8] revealed that the QIAxcel system could be considered as an effective alternative in smaller reference and regional laboratories, offering good performance. In this context, we aimed to assess the QIAxcel accuracy for allele calling of MIRU-VNTR loci in two regional reference laboratories with limited resources and where agarose gel electrophoresis is used, but with reproducibility issues and low throughput.…”

MIRU-profiler: a rapid tool for determination of 24-loci MIRU-VNTR profiles from assembled genomes ofMycobacterium tuberculosis

“…These discrepancies could be attributed to the errors in the interpretation of experimental results, microevolution or inaccuracies in the genome assembly. The experimental result is prone to errors due to sub-optimal PCR amplification, which results in multiple bands on an agarose gel electrophoresis and affects the interpretation of experimental results ( Nikolayevskyy et al, 2016 ; Supply, 2005 ). The microevolution could also result in minor discrepancies between the experimental genotyping and WGS.…”