Description of clusters containing 3 or more isolates in this study, and their worldwide distribution in the SITVIT2 database.

<p>* Worldwide distribution is reported for regions with more than 3% of a given SITs as compared to their total number in the SITVIT2 database. The definition of macro-geographical regions and sub-regions (<a href="http://unstats.un.org/unsd/methods/m49/m49regin.htm" target="_blank">http://unstats.un.org/unsd/methods/m49/m49regin.htm</a>) is according to the United Nations; Regions: AFRI (Africa), AMER (Americas), ASIA (Asia), EURO (Europe), and OCE (Oceania), subdivided in: E (Eastern), M (Middle), C (Central), N (Northern), S (Southern), SE (South-Eastern), and W (Western). Furthermore, CARIB (Caribbean) belongs to Americas, while Oceania is subdivided in 4 sub-regions, AUST (Australasia), MEL (Melanesia), MIC (Micronesia), and POLY (Polynesia). Note that in our classification scheme, Russia has been attributed a new sub-region by itself (Northern Asia) instead of including it among rest of the Eastern Europe. It reflects its geographical localization as well as due to the similarity of specific TB genotypes circulating in Russia (a majority of Beijing genotypes) with those prevalent in Central, Eastern and South-Eastern Asia.</p><p>** The 3 letter country codes are according to <a href="http://en.wikipedia.org/wiki/ISO_3166-1_alpha-3" target="_blank">http://en.wikipedia.org/wiki/ISO_3166-1_alpha-3</a>; countrywide distribution is only shown for SITs with ≥3% of a given SITs as compared to their total number in the SITVIT2 database. Note that FXX code designates Metropolitan France.</p><p>Description of clusters containing 3 or more isolates in this study, and their worldwide distribution in the SITVIT2 database.</p

A First Insight on the Population Structure of <i>Mycobacterium tuberculosis</i> Complex as Studied by Spoligotyping and MIRU-VNTRs in Santiago, Chile

<div><p>Tuberculosis (TB) remains a significant public health problem worldwide, but the ecology of the prevalent mycobacterial strains, and their transmission, can vary depending on country and region. Chile is a country with low incidence of TB, that has a geographically isolated location in relation to the rest of South American countries due to the Andes Mountains, but recent migration from neighboring countries has changed this situation. We aimed to assess the genotypic diversity of <i>Mycobacterium tuberculosis</i> complex (MTBC) strains in Santiago, Chile, and compare with reports from other Latin-American countries. We analyzed MTBC isolates from pulmonary tuberculosis cases collected between years 2008 and 2013 in Central Santiago, using two genotyping methods: spoligotyping and 12-loci mycobacterial interspersed repetitive unit-variable number of tandem repeats (MIRU-VNTRs). Data obtained were analyzed and compared to the SITVIT2 database. Mean age of the patients was 47.5 years and 61% were male; 11.6% were migrants. Of 103 strains (1 isolate/patient) included, there were 56 distinct spoligotype patterns. Of these, 16 strains (15.5%) corresponded to orphan strains in the SITVIT2 database, not previously reported. Latin American and Mediterranean (LAM) (34%) and T (33%) lineages were the most prevalent strains, followed by Haarlem lineage (16.5%). Beijing family was scarcely represented with only two cases (1.9%), one of them isolated from a Peruvian migrant. The most frequent clustered spoligotypes were SIT33/LAM3 (10.7%), SIT53/T1 (8.7%), SIT50/H3 (7.8%), and SIT37/T3 (6.8%). We conclude that LAM and T genotypes are the most prevalent genotypes of MTBC in Santiago, Chile, and together correspond to almost two thirds of analyzed strains, which is similar to strain distribution reported from other countries of Latin America. Nevertheless, the high proportion of SIT37/T3, which was rarely found in other Latin American countries, may underline a specific history or demographics of Chile related to probable human migrations and evolutions.</p></div

Description of 40 shared-types (SITs; n = 87 isolates) and corresponding spoligotyping defined lineages/sublineages starting from a total of 103 <i>M</i>. <i>tuberculosis</i> strains isolated in Santiago, Chile.

<p>* A total of 39/40 SITs containing 85 isolates matched a preexisting shared-type in the database, whereas 1/40 SIT (n = 2 isolates) was newly created. A total of 13/40 SITs containing 60 isolates were clustered within this study (2 to 11 isolates per cluster) while 27/40 SITs containing 27 strains were unique (for total unique strains, one should add to this number the 16 orphan strains, which brings the number of unclustered isolates in this study to 43/103 or 41.75%, and clustered isolates to 60/103 or 58.25%). Note that SIT followed by an asterisk indicates “newly created” SIT due to 2 or more strains belonging to an identical new pattern within this study or after a match with an orphan in the database; SIT designation followed by number of strains: 4013* this study n = 2, USA n = 1.</p><p>** Lineage designations according to SITVIT2 using revised SpolDB4 rules; “Unknown” designates patterns with signatures that do not belong to any of the major lineages described in the database.</p><p>*** Clustered strains correspond to a similar spoligotype pattern shared by 2 or more strains “within this study”; as opposed to unique strains harboring a spoligotype pattern that does not match with another strain from this study. Unique strains matching a preexisting pattern in the SITVIT2 database are classified as SITs, whereas in case of no match, they are designated as “orphan”.</p><p>Description of 40 shared-types (SITs; n = 87 isolates) and corresponding spoligotyping defined lineages/sublineages starting from a total of 103 <i>M</i>. <i>tuberculosis</i> strains isolated in Santiago, Chile.</p

A minimum spanning tree (MST) illustrating evolutionary relationships between spoligotypes and 12-loci MIRU-VNTRs in Santiago, Chile (n = 103 isolates).

<p><b>(A)</b> MST constructed on all spoligotypes; <b>(B)</b> MST constructed on all 12-loci MIRUs alone; and <b>(C)</b> MST constructed on the combination of spoligotypes and 12-loci MIRUs. The phylogenetic tree connects each genotype based on degree of changes required to go from one allele to another. The structure of the tree is represented by branches (continuous vs. dashed and dotted lines) and circles representing each individual pattern. Note that the length of the branches represents the distance between patterns while the complexity of the lines (continuous, gray dashed and gray dotted) denotes the number of allele/spacer changes between two patterns: solid lines, 1 or 2 or 3 changes (thicker ones indicate a single change, while the thinner ones indicate 2 or 3 changes); gray dashed lines represent 4 changes; and gray dotted lines represent 5 or more changes. The size of the circle is proportional to the total number of isolates in our study, illustrating unique isolates (smaller nodes) versus clustered isolates (bigger nodes). The separation inside circle also indicates the number of strains. The color of the circles indicates the phylogenetic lineage to which the specific pattern belongs. The labels of nodes indicate SITs and 12-MITs respectively in Fig. A and B; and main SIT/12-MIT couples (n ≥ 2 isolates) are represented in Fig. C.</p

Main published studies (2006–2013) reporting distribution of <i>Mycobacterium tuberculosis</i> lineages in Central and South America.

<p>Main published studies (2006–2013) reporting distribution of <i>Mycobacterium tuberculosis</i> lineages in Central and South America.</p

Distribution of main lineages according to SITVIT2 starting from a total of 103 <i>M</i>. <i>tuberculosis</i> strains isolated in Santiago, Chile.

<p>Distribution of main lineages according to SITVIT2 starting from a total of 103 <i>M</i>. <i>tuberculosis</i> strains isolated in Santiago, Chile.</p

A schematic model for the impact of the seasonality and levels of Vitamin D (VD) metabolites on the risk of being infected with <i>M</i>. <i>tuberculosis</i> and the protective mechanisms of the immune response.

<p>(<b>A</b>) During spring/summer season, sunny days favor outdoor activities, decrease close contact with people with TB and increase sun light exposure promoting enhanced skin production of 7-dehydrocholesterol (7DHC), the precursor of active VD, and the synthesis of D3 metabolites first in liver and then in kidney. The main production of 1,25-dihydroxyvitamin D3 (1,25(OH)D3) occurs in the kidney but it can also be produced by inflammatory cells during an immune response to infections [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0175400#pone.0175400.ref010" target="_blank">10</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0175400#pone.0175400.ref011" target="_blank">11</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0175400#pone.0175400.ref023" target="_blank">23</a>]. Alveolar macrophages recognize molecules associated with <i>M</i>. <i>tuberculosis</i>, such as the mycobacterial lipoprotein LpqH, through Toll-like receptors (TLRs) such as TLR2/1 and the co-receptor CD14 expressed on their cell surface [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0175400#pone.0175400.ref020" target="_blank">20</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0175400#pone.0175400.ref024" target="_blank">24</a>]. Engagement of these receptors induce a cell signaling pathway that include AMPK and p38 MAPK activation, which leads to upregulation of CYP27B1 hydroxylase and the conversion of 25OHD into 1,25(OH)D3 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0175400#pone.0175400.ref024" target="_blank">24</a>]. Given that immune cells can also express the VD receptor (VDR), 1,25(OH)D3 binds to heterodimer formed between the VDR and the retinoid X receptor (RXR) and translocate into the nucleus where this transcription factor complex specifically recognizes DNA sequences named VD response elements (VDRE) leading to the production of antimicrobial peptides, such as LL-37 (cathelicidin) and ß-defensin 2 (BD2) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0175400#pone.0175400.ref024" target="_blank">24</a>]. Cathelicidin induced by 1,25(OH)D3 drives the elimination of engulfed mycobacteria by promoting fusion of autophagosomes containing mycobacteria with lysosomes [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0175400#pone.0175400.ref024" target="_blank">24</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0175400#pone.0175400.ref025" target="_blank">25</a>]. <i>M</i>. <i>tuberculosis</i> infection and 1,25(OH)D3 induce IL-1ß gene expression, which binds via IL-1ß receptor to epithelial alveolar cells to promote the expression of BD2. The release of BD2 along with 1,25(OH)D3 contributes to control mycobacterial proliferation in the macrophage [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0175400#pone.0175400.ref026" target="_blank">26</a>]. (<b>B</b>) During autumn/winter season, cold, cloudy and rainy days promote indoor lifestyle and enhanced contact with people with TB, particularly under overcrowded living conditions. Reduced sun light exposure leads to significantly low skin production of 7DHC and consequently reduced synthesis of D3 metabolites in liver and kidney, resulting in detrimental innate immune response against <i>M</i>. <i>tuberculosis</i>. Concomitantly, reduced levels of cathelicidin impairs autophagy process. Overall, the combination of these factors would favor a higher susceptibility of being infected.</p

Regression Analysis of associations with Vitamin D levels among all TB household contacts (n = 139).

<p>Regression Analysis of associations with Vitamin D levels among all TB household contacts (n = 139).</p

Plasma levels of 25OHD and inflammatory parameters in active pulmonary TB cases, household contacts (HHC) and non-HHC subjects.

<p><b>A)</b> 25OHD levels in active pulmonary TB cases (n = 92), household contacts (HHC) with latent TB infection (QFT(+), n = 55), HHC without latent TB infection (QFT(-), n = 84) and non-HHC subjects (n = 31). <b>B)</b> Ultrasensitive C-reactive protein (CRP) in active pulmonary TB cases (n = 23), HHC with latent TB infection (QFT(+), n = 33), HHC without latent TB infection (QFT(-), n = 53) and non-HHC subjects (n = 31). <b>C)</b> Interleukin 6 (IL-6) in active pulmonary TB cases (n = 79), HHC with latent TB infection (QFT(+), n = 36), HHC without latent TB infection (QFT(-), n = 32) and non-HHC subjects (n = 19). <b>D</b>) TNF-α in active pulmonary TB cases (n = 79), HHC with latent TB infection (QFT(+), n = 36), HHC without latent TB infection (QFT(-), n = 32) and non-HHC subjects (n = 19). (*** = p< 0.001, ** = p<0.01, * = p<0.05). Horizontal line represents the median of each subject group.</p

Clinical and demographic characteristics of tuberculosis (TB) cases, household contacts (All HHC) and non-household contacts control group (Non-HHC) at enrollment.

<p>Clinical and demographic characteristics of tuberculosis (TB) cases, household contacts (All HHC) and non-household contacts control group (Non-HHC) at enrollment.</p