Description of 55 shared-types (SITs; n = 198 isolates) and corresponding spoligotyping defined lineages/sublineages starting from a total of 220 <i>M. tuberculosis</i> strains isolated in Malaysia.

a<p>Description of 55 shared-types (SITs; n = 198 isolates): 49/55 SITs containing 187 isolates matched a preexisting shared-type in the database, whereas 6/55 SITs (n = 11 isolates) were newly created. A total of 18/55 SITs containing 161 isolates were clustered within this study (2 to 56 isolates per cluster) while 37/55 SITs containing 37 strains were unique (for total unique strains, one should add to this number the 22 orphan strains, which brings the number of unclustered isolates in this study to 59/220 or 26.82%, and clustered isolates to 161/220 or 73.18%). Note that SITs followed by an asterisk indicates "newly created” SITs 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 designations followed by number of strains: 3993* this study n = 1, MYS n = 1; 3994* this study n = 2, MYS n = 1; 3994* this study n = 2; 3995* this study n = 2; 3996* this study n = 2; 3997* this study n = 2; 3998* this study n = 2.</p>b<p>Lineage designations according to SITVITWEB rules; “Unknown” designates patterns with signatures that do not belong to any of the major lineages described in the database.</p>c<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” (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114832#pone-0114832-t001" target="_blank">Table 1</a>).</p><p>Description of 55 shared-types (SITs; n = 198 isolates) and corresponding spoligotyping defined lineages/sublineages starting from a total of 220 <i>M. tuberculosis</i> strains isolated in Malaysia.</p

Customized map of phylogeographical distribution of major <i>M. tuberculosis</i> lineages in the neighboring countries of Malaysia (data extracted from the updated version of the SITVIT2 database).

<p>One may notice the similarity in the lineages distribution of this study as compared to Malaysian strains previously entered in SITVIT2. Note that the map file was downloaded under Creative Commons License using the link: <a href="http://upload.wikimedia.org/wikipedia/commons/c/c2/Blank" target="_blank">http://upload.wikimedia.org/wikipedia/commons/c/c2/Blank</a> Map Pacific World.svg and was manually modified for representative purposes only.</p

Description of clusters containing >1% (n = 3 or more) isolates in this study and their worldwide distribution in the SITVIT2 database.

a<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>b<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.</p><p>Description of clusters containing >1% (n = 3 or more) isolates in this study and their worldwide distribution in the SITVIT2 database.</p

Phylogenetical trees illustrating evolutionary relationships between <i>M. tuberculosis</i> spoligotypes in Malaysia.

<p>(A) Spoligoforest tree drawn using the SpolTools software (available through <a href="http://www.emi.unsw.edu.au/spolTools" target="_blank">http://www.emi.unsw.edu.au/spolTools</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114832#pone.0114832-Tang1" target="_blank">[14]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114832#pone.0114832-Reyes1" target="_blank">[15]</a>), and shown as Fruchterman Reingold tree. The Figure was drawn on all patterns including orphan patterns (n = 220). In both cases, each spoligotype pattern from the study is represented by a node with area size being proportional to the total number of isolates with that specific pattern. Changes (loss of spacers) are represented by directed edges between nodes, with the arrowheads pointing to descendant spoligotypes. In this representation, the heuristic used selects a single inbound edge with a maximum weight using a Zipf model. Solid black lines link patterns that are very similar, i.e., loss of one spacer only (maximum weigh being 1.0), while dashed lines represent links of weight comprised between 0.5 and 1, and dotted lines a weight less than 0.5. One may notice the predominance of SIT1/Beijing (n = 56), SIT745/EAI1-SOM (n = 33), SIT591/EAI6-BGD1 (n = 13), SIT256/EAI5 (n = 12), and SIT236/EAI5 (n = 10). Furthermore, note the phylogeographical specificity of SIT745 for Malaysia, and the fact that it seems to have not largely spread in other countries/regions (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114832#pone-0114832-t003" target="_blank">Table 3</a>). (B) A minimum spanning tree (MST) illustrating evolutionary relationships between M. tuberculosis spoligotypes. MST constructed on all isolates including the orphan patterns (n = 220). 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 color of the circles indicates the phylogenetic lineage to which the specific pattern belongs. Note that orphan patterns are circled in orange (22 strains belonged to orphans in our study). Note that the colors used are the same in Spoligoforest tree and MST.</p

Zoomed part of Spoligoforest tree showing SIT745 tentatively relabeled "EAI1-MYS" and its spoligotype descendants, along with their binary spoligotyping descriptions highlighting the specific absence of spacers 37, 38 and 40.

<p>Zoomed part of Spoligoforest tree showing SIT745 tentatively relabeled "EAI1-MYS" and its spoligotype descendants, along with their binary spoligotyping descriptions highlighting the specific absence of spacers 37, 38 and 40.</p