A two dimensional scatter plot based on principal component analysis (PCA) based on 15 MIRU-VNTR individual alleles.

<p>MIRU-VNTR marker having similarity in MIRU-VNTR allelic tend to clusters together.</p

Evaluation of spoligotyping, SNPs and customised MIRU-VNTR combination for genotyping <i>Mycobacterium tuberculosis</i> clinical isolates in Madagascar

<div><p>Background</p><p>Combining different molecular typing methods for <i>Mycobacterium tuberculosis</i> 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.</p><p>Method</p><p>Spoligotyping 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.</p><p>Results</p><p>Of 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.</p><p>Conclusion</p><p>According 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.</p></div

Schematic overview of the typing scheme developed to differentiate MTBC clinical isolates studied.

<p>SNPs: Single Nucleotide Polymorphism. The blue arrow indicated that the discrimination level increased with the number of markers typed.</p

Discrimination power of MIRU-VNTR genetic marker combination proposed.

<p>Discrimination power of MIRU-VNTR genetic marker combination proposed.</p

Global HGDI and HGDI of each locus for spoligotyping family.

<p>Global HGDI and HGDI of each locus for spoligotyping family.</p

Additional file 1: Table S1. of Assessment of tuberculosis spatial hotspot areas in Antananarivo, Madagascar, by combining spatial analysis and genotyping

Distribution of the 88 spoligotypes obtained with the 394 typed patients. (DOCX 38 kb

Peripheral blood gene expression as a function of ESAT-6 ELISPOT response in the clinical groups.

<p>(A) <i>TNFR1</i>, (B) <i>TNFR2</i>, (C) <i>FLIPs</i>, (D) <i>FLICE</i>. The data shown are the median and ranges of mRNA levels normalized and expressed as a number of copies per 10⁵ copies of mRNA for the housekeeping gene, <i>HuPO</i>. Significant differences in gene expression between clinical groups are indicated.</p

Comparison of peripheral blood gene expression in the contacts and controls after 3 months of follow-up.

<p>Expression on inclusion in the study (M0) and after 3 months of follow-up (M3) for (A) <i>TNFR1</i>, (B) <i>TNFR2</i>, (C) <i>FLIPs</i> and (D) FLICE. The data shown are the median and ranges for mRNA levels normalized and expressed as the number of copies per 10⁵ copies of the mRNA for the housekeeping gene, <i>HuPO</i>. Mann-Whitney U tests were used for the pairwise comparison of groups. Significant differences between the testing periods are shown. HC: household contact, CC: community control.</p

Sequences of the primers and probes used to quantify gene expression by real-time PCR.

<p> <b>L: Left primer (5′--------3′).</b></p><p> <b>R: Right primer (5′--------3′).</b></p><p> <b>P: Probe (5′ FAM--------TAMRA 3′).</b></p

Peripheral blood cell distribution as a function of the presence or absence of TB symptoms.

<p>(A) Monocytes, (B) neutrophils and (C) lymphocytes. The data shown are the mean cell percentage+SD. Significant differences in gene expressions between clinical groups are indicated. TB, index cases and household contacts presenting TB symptoms.</p