Evaluation of the new advanced 15-loci MIRU-VNTR genotyping tool in Mycobacterium tuberculosis molecular epidemiology studies

Abstract

Background: During the last few years, PCR-based methods have been developed to simplify and reduce the time required for genotyping Mycobacterium tuberculosis (MTB) by standard approaches based on IS6110-Restriction Fragment Length Polymorphism (RFLP). Of these, MIRU-12-VNTR (Mycobacterial interspersed repetitive units- variable number of tandem repeats) (MIRU-12) has been considered a good alternative. Nevertheless, some limitations and discrepancies with RFLP, which are minimized if the technique is complemented with spoligotyping, have been found. Recently, a new version of MIRU-VNTR targeting 15 loci (MIRU-15) has been proposed to improve the MIRU-12 format.

Results: We evaluated the new MIRU-15 tool in two different samples. First, we analyzed the same convenience sample that had been used to evaluate MIRU-12 in a previous study, and the new 15-loci version offered higher discriminatory power (Hunter-Gaston discriminatory index [HGDI]: 0.995 vs 0.978; 34.4% of clustered cases vs 57.5%) and better correlation (full or high correlation with RFLP for 82% of the clusters vs 47%). Second, we evaluated MIRU-15 on a population-based sample and, once again, good correlation with the RFLP clustering data was observed (for 83% of the RFLP clusters). To understand the meaning of the discrepancies still found between MIRU-15 and RFLP, we analyzed the epidemiological data for the clustered patients. In most cases, splitting of RFLP-clustered patients by MIRU-15 occurred for those without epidemiological links, and RFLP-clustered patients with epidemiological links were also clustered by MIRU-15, suggesting a good epidemiological background for clustering defined by MIRU-15.

Conclusion: The data obtained by MIRU-15 suggest that the new design is very efficient at assigning clusters confirmed by epidemiological data. If we add this to the speed with which it provides results, MIRU-15 could be considered a suitable tool for real-time genotyping.

Figure 1

Hunter-Gaston discriminatory index (HGDI) for the loci in MIRU-12 and MIRU-15 sets. The HGDI of each locus was calculated based on the convenience sample of 134 isolates.

Figure 2

Comparative analysis between RFLP and MIRU-15 in the convenience sample. N indicates the number of isolates clustered by RFLP or MIRU. The FITS column indicates the number of isolates grouped both by RFLP and MIRU-15. The Differences column indicates the number of isolates clustered by MIRU and unclustered by RFLP (+N) or the number of isolates clustered by RFLP and unclustered by MIRU (-N). The Correlation column specifies whether correlation is Full Correlation (FC), High Correlation (HC) or No Correlation (NC). For the isolates clustered with high correlation, the single locus variation (SLV) or percentage of IS6110 band similarity is specified.

Figure 3

Detailed analysis of the discrepant cases in the No Correlation (NC) clusters. RFLP clusters are defined as Rn. M7: isolates grouped by MIRU together with the two isolates grouped in cluster R7. "A" indicates the cluster defined by MIRU but not by RFLP. N indicates the number of clustered isolates: The number of isolates clustered by MIRU and unclustered by RFLP (+N), or the number of isolates clustered by RFLP and unclustered by MIRU (-N) are highlighted in bold. MIRU loci showing differences are boxed. Spoligotypes are shown using octal code; differences are highlighted in bold.

Figure 4

Discrepancies between RFLP clustered cases and MIRU-15 data in the population sample. MIRU loci showing differences are boxed. The last column shows the epidemiological evaluation of the clustered cases. Cluster 217 was subdivided by MIRU-15 into three different MIRU types (marked with numbers 1, 2, and 3).