The Genotypic Population Structure of <em>Mycobacterium tuberculosis</em> Complex from Moroccan Patients Reveals a Predominance of Euro-American Lineages

<div><h3>Background</h3><p>Tuberculosis (TB) remains a major health problem in Morocco. Characterization of circulating <em>Mycobacterium tuberculosis</em> genotypic lineages, important to understand the dynamic of the disease, was hereby addressed for the first time at a national level.</p> <h3>Methodology/Principal Findings</h3><p>Spoligotyping was performed on a panel of 592 <em>M. tuberculosis</em> complex strains covering a 2-year period (2004–2006). It identified 129 patterns: 105 (n = 568 strains) corresponded to a SIT number in the SITVIT2 database, while 24 patterns were labeled as orphan. A total of 523 (88.3%) strains were clustered vs. 69 or 11.7% unclustered. Classification of strains within 3 large phylogenetical groups was as follows: group 1– ancestral/TbD1+/PGG1 (EAI, Bovis, Africanum), group 2– modern/TbD1−/PGG1 group (Beijing, CAS), group 3– evolutionary recent/TbD1−/PGG2/3 (Haarlem, X, S, T, LAM; alternatively designated as the Euro-American lineage). As opposed to group 3 strains (namely LAM, Haarlem, and T) that predominated (86.5% of all isolates), 6 strains belonged to group 2 (Beijing n = 5, CAS n = 1), and 3 strains (BOV_1 n = 2, BOV_4-CAPRAE) belonged to ancestral group 1 (EAI and AFRI lineage strains were absent). 12-loci MIRU-VNTR typing of the Casablanca subgroup (n = 114 strains) identified 71 patterns: 48 MITs and 23 orphan patterns; it allowed to reduce the clustering rate from 72.8% to 29.8% and the recent transmission rate from 64% to 20.2%.</p> <h3>Conclusion</h3><p>The <em>M. tuberculosis</em> population structure in Morocco is highly homogeneous, and is characterized by the predominance of the Euro-American lineages, namely LAM, Haarlem, and T, which belong to the “evolutionary recent” TbD1−/PGG2/3 phylogenetic group. The combination of spoligotyping and MIRUs decreased the clustering rate significantly, and should now be systematically applied in larger studies. The methods used in this study appear well suited to monitor the <em>M. tuberculosis</em> population structure for an enhanced TB management program in Morocco.</p> </div

Demographic and epidemiological data of the studied population from Morocco (n = 592).

a<p>Administrative regions (and cities) of origin for the 592 patients in the present study.</p>b<p>Male to Female sex ratio.</p>c<p>Significantly lower than other regions of Morocco (p<0.01).</p>d<p>NA: Not Applicable since only 1 patient originated from this region.</p

Geographical distribution of genotypic lineages of <i>M. tuberculosis</i> isolates from Morocco among ten distinct administrative regions (n = 586/592 strains; 6 strains from 3 cities were not shown as pie charts due to their low numbers: Oujda n = 1; Inezgane n = 2; Ouarzazate n = 3).

<p>Asterisk (*) denotes the administrative region of Rabat-Salé-Zemmour-Zaër). The references to color and lineages in this figure are as follows: (i) <u>Haarlem1</u>: SITs 47, 62, 143, 218, 609, 1139, 1155, 2333, 2567, 2 orphans; (ii) <u>Haarlem3</u>: SITs 36, 49, 50, 433, 741, 1135, 1539, 2338, 3077, 3078; (iii) <u>Other Haarlem genotypes</u>: Haarlem2 (SIT2), and Haarlem (1 orphan); (iv) <u>LAM3</u>: SITs 33, 105, 106; (v) <u>LAM4</u>: SIT60, 163, 1 orphan; (vi) <u>LAM9</u>: SITs 42, 161, 162, 177, 492, 731, 737, 770, 822, 866, 1070, 1074, 1075, 1154, 1277, 1832, 1 orphan; (vii) <u>Other LAM genotypes</u>: LAM1 (SITs 964,2372), LAM5 (SITs 93, 1072, 1999,2341), LAM6 (SIT64), LAM10-CAM (SIT61), LAM12-Madrid (SIT209), LAM (SITs 1537,3075, 7 orphans); (viii) <u>S lineage</u>: SIT34, 466, 784, 2516; (ix) <u>ill-defined T</u>: SIT7, 31, 32, 37, 40, 44, 53, 58, 65, 73, 77, 78, 102, 120,164, 244, 291, 334, 373, 751, 926, 1069, 1105, 1580,1626, 1655, 2025, 3073, 3076, 6 orphans; (v) and <u>Other/undefined</u>: these included Beijing (SIT1), CAS1-Delhi (SIT26), BOV_1 (SIT481, 1026), BOV_4-CAPRAE (SIT644), as well as other undefined profiles marked as Unk (unknown) in <b><u><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047113#pone.0047113.s001" target="_blank">Table S1</a></u></b>.</p

Description of predominant shared-types found 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 ≥5% of a given SITs as compared to their total number in the SITVIT2 database.</p

Minimum spanning tree (MST) showing evolutionary relationships among spoligotypes and lineages observed in Morocco.

<p>The length of the branches indicates the level of changes induced by loss or gain of spoligotype spacers to induce a shift from one allele to another (solid lines, single or 2 spacers changes; dotted lines, 3 or more spacer changes; color of circles shows number of isolates : sky-blue, 1 to 2 isolates; marine blue, 3 to 5 isolates; deep blue, 6 to 10 strains; brown, 11 to 20 strains; and red, >20 strains). Predominant genotypic lineages, sublineages and subsets are shown in distinct colors as follows: Haarlem (subdivided in Haarlem 1 and 3), LAM (subdivided in LAM subsets 1 and 2), S, and T (please refer to text for a detailed description).</p

Knowledge of patients about TB and its treatment.

<p>Knowledge of patients about TB and its treatment.</p

Risk Factors associated with TB treatment default and level of significance in multivariable logistic regression model.

<p>(*) Unadjusted odds ratio (univariate analysis).</p><p>(**) Adjusted odds ratio (multivariable analysis).</p

Comparison of characteristics of patients who defaulted from TB treatment (cases) versus patients who completed TB treatment (controls): results of univariate analyses.

<p>**n = 268, 6 controls and 3 cases declined to respond to this question; median urban income in Morocco was 6,100 dirhams/month in 2009.</p

ROC curves for different point cut-offs for the proposed survey tool.

<p>The area under the ROC curve (AUC) for each score and its 95% confidence interval are provided.</p

Organization of risk factors for default in urban Morocco within the information-motivation-behavioral (IMB) skills theoretical framework.

<p>*Statistically significant in multivariate analyses in this study.</p