Evaluation of spoligotyping, SNPs and customised MIRU-VNTR combination for genotyping Mycobacterium tuberculosis clinical isolates in Madagascar.

Niaina Rakotosamimanana,Christophe Sola,Rondroarivelo Rasoahanitralisoa,Olivier Neyrolles,Sebastien Gagneux,David Stucki,Voahangy Rasolofo Razanamparany

doi:10.1371/journal.pone.0186088

Abstract

BackgroundCombining different molecular typing methods for Mycobacterium tuberculosis complex (MTBC) can be a powerful tool for molecular epidemiology-based investigation of TB. However, the current standard method that provides high discriminatory power for such a combination, mycobacterial interspersed repetitive units-variable numbers of tandem repeats typing (MIRU-VNTR), is laborious, time-consuming and often too costly for many resource-limited laboratories. We aimed to evaluate a reduced set of loci for MIRU-VNTR typing in combination with spoligotyping and SNP-typing for routine molecular epidemiology of TB.MethodSpoligotyping and SNP-typing, in combination with the 15 loci MIRU-VNTR typing, were first used to type clinical MTBC isolates (n = 158) from Madagascar. A step by step reduction of MIRU-VNTR loci number was then performed according to the Hunter and Gaston Discriminatory Index (HGDI) and to the Principal component analysis (PCA) correlation with the spoligotype profiles to evaluate the discrimination power inside the generated spoligotype clusters. The 15 MIRU-VNTR was used as reference and SNP-typing was used to determine the main MTBC lineages.ResultsOf the 158 clinical isolates studied, the SNP-typing classified 23 into Lineage 1 (14.6%), 31 into Lineage 2 (19.6%), 23 into Lineage 3 (14.6%) and 81 into Lineage 4 strains (51.3%). 37 different spoligotypes profiles were obtained, 15 of which were unique and 20 in clusters. 15-loci MIRU-VNTR typing revealed 144 different genotypes: 132 isolates had a unique MIRU-VNTR profile and 27 isolates were grouped into 12 clusters. After a stepwise reduction of the MIRU-VNTR loci number within each main spoligotype families, three different sets composed of 5 customised MIRU-VNTR loci had a similar discrimination level to the reference 15 loci MIRU-VNTR in lineage 1, lineage 2 and lineage 3. For lineage 4, a set of 4 and 3 MIRU-VNTR loci were proposed to subtype the Harleem and LAM spoligotype families, respectively. For the T spoligotype family, a set of 5 MIRU-VNTR loci was proposed.ConclusionAccording to the lineages and the spoligotype families, the number of MIRU-VNTR loci can be reduced to get an optimal classification of MTBC. These customized sets of MIRU-VNTR loci reduce workload and save resources while maintaining optimal discriminatory power.

Highlights

M. tuberculosis is a widespread global human pathogen whose infection causes an estimated 10.4 million cases of tuberculosis (TB) and 1.8 million deaths every year [1]
According to the lineages and the spoligotype families, the number of MIRU-VNTR loci can be reduced to get an optimal classification of Mycobacterium tuberculosis complex (MTBC)
SNP-typing, spoligotyping, and MIRU-VNTR typing are three PCR-based typing method used for molecular epidemiology of MTBC strains

Summary

Introduction

M. tuberculosis is a widespread global human pathogen whose infection causes an estimated 10.4 million cases of tuberculosis (TB) and 1.8 million deaths every year [1]. SNP-typing, spoligotyping, and MIRU-VNTR typing are three PCR-based typing method used for molecular epidemiology of MTBC strains. Spoligotyping reveals the absence or presence of unique spacer sequences located between the direct-repeat (DR) sequences of the Clustered Regularly Interspersed Short Palindromic Repeat (CRISPR) region of MTBC genomes [8]. This method is a membrane-based reverse-line blotting procedure, recently adapted to a highthroughput microbead-based format, whose digital results ease the exchange of data between laboratories [9].

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