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Original article
Mycobacterium tuberculosis Beijing in the State of Nuevo Leon, Mexico
Mycobacterium tuberculosis Beijing en el Estado de Nuevo León, México
Nohemí Catana-Botelloa, Pola Becerril-Montesb,
Corresponding author
pbecerril@hotmail.com

Corresponding authors.
, Jorge Castro-Garzac, Francisco González-Salazarb,d,
Corresponding author
fgonz75@hotmail.com

Corresponding authors.
, Horacio Almanza-Reyese,
Corresponding author
almanzareyes@hotmail.com

Corresponding authors.
, María De Los Ángeles Del Bosque-Moncayof, Alejandro Morales-Vargasf, Víctor Manuel Velázquez-Morenof
a Faculty of Biological Sciences, Autonomous University of Nuevo León, Ave. Pedro de Alba S/N, Niños Héroes, Ciudad Universitaria, San Nicolás de los Garza, Nuevo León 64260, Mexico
b Biomedical Research Center of the Northeast, Mexican Social Security Institute, San Luis Potosí 611, Col. Independencia, Monterrey, Nuevo León 64720, Mexico
c Secretary of Health of the State of Nuevo León, C. Mariano Matamoros 520, Nuevo León 64000, Mexico
d Health Sciences, University of Monterrey, Av. Ignacio Morones Prieto 4500 poniente, Col. Jesús M. Garza, San Pedro Garza García, Nuevo León 66238, Mexico
e Faculty of Medicine and Psychology, Autonomous University of Baja California, Universidad 14418, UABC, Parque Internacional Industrial Tijuana, Tijuana Baja California 22390, Mexico
f State Public Health Laboratory of Nuevo León, Prof. Serafín Peña 2211, Valles de La Silla, Guadalupe, Nuevo León 67180, Mexico
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        "titulo" => "<span class="elsevierStyleItalic">Mycobacterium tuberculosis</span> Beijing en el Estado de Nuevo Le&#243;n&#44; M&#233;xico"
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    "textoCompleto" => "<span class="elsevierStyleSections"><span id="sec0005" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0030">Introduction</span><p id="par0025" class="elsevierStylePara elsevierViewall">Since <span class="elsevierStyleItalic">Mycobacterium tuberculosis</span> genotype Beijing was found as the causative agent of the drug-resistant tuberculosis outbreak in the 1990s&#44; there was increased interest to know the origin&#44; epidemiology&#44; and specific characteristics of the Beijing strain&#44; which appears to have higher virulence and greater spread ability&#46; <span class="elsevierStyleItalic">M&#46; tuberculosis</span> lineage 2&#44; known as the Beijing lineage&#44; is particularly abundant in Asian countries&#44;<a class="elsevierStyleCrossRef" href="#bib0275"><span class="elsevierStyleSup">27</span></a> and is considered a clonal population derived from a common ancestor&#44; spreading radially from Beijing to other geographic areas&#46;<a class="elsevierStyleCrossRef" href="#bib0220"><span class="elsevierStyleSup">16</span></a> Several reports show that Beijing strains are now distributed worldwide&#44; and are often associated with multidrug resistance&#46;<a class="elsevierStyleCrossRefs" href="#bib0150"><span class="elsevierStyleSup">2&#44;11</span></a> The higher resistance characteristic of these strains is possibly due to an increased rate of mutation&#46;<a class="elsevierStyleCrossRefs" href="#bib0180"><span class="elsevierStyleSup">8&#44;12</span></a></p><p id="par0030" class="elsevierStylePara elsevierViewall">A minor sublineage&#44; known as variant 0&#44; belonging to a branch of the Beijing family&#44; caused an outbreak in HIV&#43; patients in penitentiaries and hospitals at New York City in the 1990s and was designated as the W strain&#46; Similarly to this case&#44; there are several variants around the world &#40;Bifani 2321&#44; Kurepina 31&#41;&#46;</p><p id="par0035" class="elsevierStylePara elsevierViewall">Diverse animal models have shown that Beijing strains are more virulent&#44; have a higher growth rate&#44; produce more histopathological changes&#44; and more deaths than other strains&#46; They have special characteristics in their protein and lipid structures and in the way they interact with the immune system&#46; Moreover&#44; this group has been associated with specific polymorphisms in human genes related to the immune response suggesting a co-evolution with humans&#46;<a class="elsevierStyleCrossRef" href="#bib0235"><span class="elsevierStyleSup">19</span></a></p><p id="par0040" class="elsevierStylePara elsevierViewall">However&#44; the heterogeneity of these organisms in terms of drug-resistance&#44; virulence &#40;Dormans 137&#41;&#44; and epidemiological characteristics suggests a genetic diversity leading to several sublineages&#46;<a class="elsevierStyleCrossRef" href="#bib0240"><span class="elsevierStyleSup">20</span></a> This diversity has been related to the fact that the dissemination of this genotype from its original focus radiated worldwide in several waves&#44; mainly in the 19th century&#59; and the increased drug resistance came as a result of the weakness of health systems&#44; indiscriminate use of antibiotics&#44; the emergence of the HIV&#47;AIDS epidemic and an increase in the pathogen&#39;s population size&#46;<a class="elsevierStyleCrossRef" href="#bib0220"><span class="elsevierStyleSup">16</span></a></p><p id="par0045" class="elsevierStylePara elsevierViewall">Based on the presence or absence of the <span class="elsevierStyleItalic">IS6110</span> sequence in the NTF &#40;noise transfer function&#41; region&#44; the Beijing strains are classified into modern or typical strains and ancestral or atypical strains&#46;<a class="elsevierStyleCrossRef" href="#bib0145"><span class="elsevierStyleSup">1</span></a> Among the modern strains are those containing one <span class="elsevierStyleItalic">IS6110</span> sequence in the NTF region and the W strain with a double insertion&#44; while the ancestral strains do not have these insertions&#46;<a class="elsevierStyleCrossRef" href="#bib0225"><span class="elsevierStyleSup">17</span></a> Large sequence polymorphism &#40;LSPs&#41; analysis identified differences among Beijing strains&#46;<a class="elsevierStyleCrossRef" href="#bib0265"><span class="elsevierStyleSup">25</span></a> The RD105 and RD207 regions are absent in all the Beijing family&#44; whereas the RD142&#44; RD150 and RD181 regions divide the family into 4 monophyletic groups&#46;<a class="elsevierStyleCrossRefs" href="#bib0185"><span class="elsevierStyleSup">9&#44;25</span></a> The RD181 deletion occurred early during the evolution of the lineage&#44; while the RD150 and RD142 deletions have a low value for the phylogeny&#46;<a class="elsevierStyleCrossRef" href="#bib0170"><span class="elsevierStyleSup">6</span></a> The relationship between the members of the Beijing family&#44; the emergence of drug resistance&#44; the increased virulence&#44; and their spreading ability justify the effort to characterize the isolates to promptly detect the presence of high-risk variants that could potentially affect individuals&#46; In this study&#44; spoligotyping and 18-locus MIRU-VNTR methods were used to identify different strains of <span class="elsevierStyleItalic">Mycobacterium Beijing</span> isolated from humans&#44; with the aim to better analyze genetic diversity and determine their dominant types in the State of Nuevo Leon&#44; Mexico&#46;</p></span><span id="sec0010" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0035">Materials and methods</span><span id="sec0015" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0040">Sampling</span><p id="par0050" class="elsevierStylePara elsevierViewall">In this study&#44; 406 isolates of <span class="elsevierStyleItalic">M&#46; tuberculosis</span> recovered from sputum samples of patients diagnosed with pulmonary tuberculosis were analyzed by using the spoligotyping technique&#46;</p></span><span id="sec0020" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0045">Ethical approval</span><p id="par0055" class="elsevierStylePara elsevierViewall">For this study&#44; all experiments were performed in accordance with the Declaration of Helsinki&#46; Informed consent was obtained from all participants and ethical approval was obtained from the Research Ethics Committee of the Faculty of Medicine and Psychology of the Autonomous University of Baja California &#40;ref&#46;&#58; 2046&#41;&#46;</p><p id="par0060" class="elsevierStylePara elsevierViewall">All of them were isolated in the State of Nuevo Leon&#44; Mexico from 2008 to 2015&#46; Samples were processed using the Petroff method and seeded into L&#246;wenstein&#8211;Jensen medium and MGIT liquid medium&#46; All mycobacteria were characterized by conventional tests &#40;niacin&#44; catalase&#44; and nitrates or immunochromatography&#41; at the State Public Health Laboratory of Nuevo Leon&#44; Mexico&#46; Genomic DNA was obtained from those isolates identified as <span class="elsevierStyleItalic">M&#46; tuberculosis</span>&#46; Two small loopfuls of mycobacterial culture were suspended in 1<span class="elsevierStyleHsp" style=""></span>ml of lysis solution &#40;Promega&#41; and heated at 80<span class="elsevierStyleHsp" style=""></span>&#176;C and then cells were lysed using a Savant Bio 101 FastPrep FP120 cell disruption system &#40;Gemini Lab&#46;Apeldoorn&#44; The Netherlands&#41; by applying five cycles of 45<span class="elsevierStyleHsp" style=""></span>s at a speed setting 6 with 2-min rest periods on ice in between runs&#46; Mycobacterial DNA was isolated using the Wizard SV Genomic DNA Purification System A2365 &#40;Promega&#41;&#44; following the manufacturer&#39;s instructions&#46; The isolated DNA was stored in 50<span class="elsevierStyleHsp" style=""></span>&#956;l of sterile nuclease-free water at 4<span class="elsevierStyleHsp" style=""></span>&#176;C until its use&#46; DNA quality was evaluated by gel electrophoresis and concentration was measured from 1<span class="elsevierStyleHsp" style=""></span>&#956;l of the resuspended samples on a Nanodrop equipment &#40;Thermo Scientific&#44; Massachusetts&#44; United State&#41;&#46;</p></span><span id="sec0025" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0050">Spoligotyping</span><p id="par0065" class="elsevierStylePara elsevierViewall">Spoligotyping of all isolates was performed by amplifying the DR region from a 20<span class="elsevierStyleHsp" style=""></span>ng&#47;50<span class="elsevierStyleHsp" style=""></span>&#956;l dilution of extracted DNA&#44; in nuclease-free water&#44; using the standard method described by Kamerbeek et al&#46;<a class="elsevierStyleCrossRef" href="#bib0210"><span class="elsevierStyleSup">14</span></a> PCR amplicons were subjected to inverse hybridization analysis &#40;<a class="elsevierStyleCrossRef" href="#sec0090">Supplementary data &#40;Table S1&#41;</a>&#41;&#44; using a commercial membrane &#40;Mapmygenome&#44; India&#41; that included spacer probes 1&#8211;43&#46;<a class="elsevierStyleCrossRef" href="#bib0210"><span class="elsevierStyleSup">14</span></a> Hybridation profile was revealed by chimioluminiscence with the ECL system &#40;Enhanced Chemiluminescent detection&#41; &#40;Amersham Biosciences&#44; Buckinghamshire&#44; United Kingdom&#41;&#46; <span class="elsevierStyleItalic">M&#46; tuberculosis</span> and <span class="elsevierStyleItalic">Mycobacterium bovis</span> BCG were the controls for each experiment&#46; The obtained profiles were compared with the spoligotyping international database SpolDB4&#46;0&#46; The strains selected for this work were those exhibiting a spoligotyping profile belonging to the Beijing family and having the RD181 deletion&#46;</p></span><span id="sec0030" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0055">Drug susceptibility test</span><p id="par0070" class="elsevierStylePara elsevierViewall">Tests were performed in a Bactec 960 equipment&#44; following the manufacturer&#39;s instructions&#46; Resistant and sensitive strains for each drug were included in each test as controls&#46;</p></span><span id="sec0035" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0060">Multiple loci VNTR analysis &#40;MLVA&#41;</span><p id="par0075" class="elsevierStylePara elsevierViewall">Eighteen chromosomal regions containing tandem repeats were amplified by PCR&#46;<a class="elsevierStyleCrossRefs" href="#bib0260"><span class="elsevierStyleSup">24&#44;23</span></a> Primers for the 18 MIRU-VNTR loci were used&#44; including twelve standard primers and six additional primers for the characterization of the Beijing genotype &#40;<a class="elsevierStyleCrossRef" href="#sec0090">Supplementary data &#40;Table S2&#41;</a>&#41;&#46; The amplicons were analyzed in 2&#37; agarose gel electrophoresis&#46; The number of repeats according to their size against a MW marker &#40;100<span class="elsevierStyleHsp" style=""></span>pb ladder&#44; Promega&#44; lugar&#41; were obtained for each locus and then a code of 18 numbers was generated and analyzed using Bionumerics v7 software &#40;Applied Maths NV&#44; Sint-Martens-Latem&#44; B&#233;lgica&#41; and a minimum spanning tree &#40;MST&#41; was constructed to identify the main lineages&#46;</p></span><span id="sec0040" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0065">Single nucleotide polymorphism &#40;SNPs&#41; assay</span><p id="par0080" class="elsevierStylePara elsevierViewall"><span class="elsevierStyleItalic">katG</span> and <span class="elsevierStyleItalic">gyrA</span> genes were analyzed by sequencing PCR fragments that were amplified from these specific regions using the primers shown in <a class="elsevierStyleCrossRef" href="#sec0090">Supplementary data &#40;Table S3</a>&#41;&#46; Sequences were aligned with the software BioEdit to identify SNPs described by Sreevatsan &#40;1977&#41;&#46;</p></span><span id="sec0045" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0070">Analysis of pks15&#47;1 presence</span><p id="par0085" class="elsevierStylePara elsevierViewall">A 144-pb segment of gene <span class="elsevierStyleItalic">pks15&#47;1</span> amplified by PCR was sequenced using the Sanger method &#40;1977&#41; and the resulting sequence was aligned using the Mega software &#40;available online <a href="https://www.megasoftware.net/">https&#58;&#47;&#47;www&#46;megasoftware&#46;net</a>&#41; and the Muscle algorithm to identify the presence of a specific 7-bp sequence characteristic of the East Asian lineage&#46;</p><span id="sec0050" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0075">Deletion of the TbD1 gene</span><p id="par0090" class="elsevierStylePara elsevierViewall">A nested PCR to amplify a 2000-bp region of gene TbD1 was conducted according to Brosch et al&#46; &#40;2002&#41;<a class="elsevierStyleCrossRef" href="#bib0155"><span class="elsevierStyleSup">3</span></a> and the resulting products were visualized on a 2&#37; agarose gel &#40;<a class="elsevierStyleCrossRef" href="#sec0090">Supplementary data &#40;Table S4&#41;</a>&#41;&#46;</p></span></span><span id="sec0055" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0080">Large sequence polymorphism &#40;LSPs&#41; analysis</span><p id="par0095" class="elsevierStylePara elsevierViewall">The presence of large sequence polymorphisms in regions RD105&#44; 181&#44; 207&#44; 150 and 142 was determined by Real Time PCR on an AB 7500 equipment &#40;Applied Biosystem&#41;&#46;</p></span><span id="sec0060" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0085">Analysis of the noise transfer function &#40;NTF&#41; region</span><p id="par0100" class="elsevierStylePara elsevierViewall">The analysis of the NTF region was performed by PCR amplification&#44; using the primers described by Plikaytis &#40;1994&#41; and modified by Wada &#40;2009&#41;&#46; The resulting PCR products were visualized on a 2&#37; agarose gel &#40;<a class="elsevierStyleCrossRef" href="#sec0090">Supplementary data &#40;Tables S5 and S6</a>&#41;&#41;&#46;</p></span></span><span id="sec0065" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0090">Results</span><p id="par0105" class="elsevierStylePara elsevierViewall">Based on the spoligotyping analysis&#44; we found fourteen strains belonging to the Beijing genotype&#44; which were characterized phylogenetically&#46; A group of 10 strains isolated from patients attended in the State of Baja California&#44; and previously identified as Beijing&#44; were included in this analysis to compare the profiles &#40;<a class="elsevierStyleCrossRef" href="#tbl0005">Table 1</a>&#41;&#46; The percentages of the most frequently found genotypes were as follows&#58; 38&#46;9&#37; belonged to the T lineage&#44; 11&#46;3&#37; to the LAM lineage&#44; 25&#46;2&#37; to the X lineage&#44; 11&#37; to the H lineage&#44; 1&#37; to the EAI2-Manila family&#44; 2&#46;9&#37; to the S lineage and 0&#46;6&#37; to the U lineage&#46; The remaining he strains belonged to 15 genotypes that were not identified in the database&#46;</p><elsevierMultimedia ident="tbl0005"></elsevierMultimedia><p id="par0110" class="elsevierStylePara elsevierViewall">The MVLA analysis showed profile variations between Beijing and non-Beijing strains&#46; Loci Mtub21&#44; QUB11b y QUB18 revealed differences in Beijing and non-Beijing &#40;123&#44; 391 and H37Rv&#41; strains&#46; Non-Beijing strains had fewer repeats&#46; MIRUs exhibited a higher discriminatory power and therefore the biggest allelic diversity among Beijing strains was observed in MIRU10&#44; MIRU31&#44; MIRU39&#44; MRU40&#44; and MIRU 26&#46; Those lacking discriminatory power were MIRU 2&#44; MIRU 10&#44; MIRU 20&#44; MIRU 23 Y MIRU 24 &#40;<a class="elsevierStyleCrossRef" href="#tbl0010">Table 2</a>&#41;&#46;</p><elsevierMultimedia ident="tbl0010"></elsevierMultimedia><p id="par0115" class="elsevierStylePara elsevierViewall">The minimum spanning tree &#40;MST&#41; showed that 79&#37; &#40;19&#41; of the strains are related&#46; In <a class="elsevierStyleCrossRef" href="#fig0005">Figure 1</a>&#44; strains belonging to the same cluster are connected by a black line and are part of a clonal complex&#46; The first cluster comprises strains 593&#44; 710 y 129&#59; a second cluster includes strains 2&#44; 22&#44; 27&#44; 45 and 53&#59; and a third cluster strains 349&#44; 362&#44; 376&#44; 115 and 524&#46; Twenty-one percent &#40;five&#41; are unrelated strains &#40;25&#44; 394&#44; 110&#44; 130 and 123&#41; and are connected by a dotted line&#46; All Beijing strains had the deletion of region TbD1&#44; characteristic of modern strains&#46;<a class="elsevierStyleCrossRef" href="#bib0155"><span class="elsevierStyleSup">3</span></a></p><elsevierMultimedia ident="fig0005"></elsevierMultimedia><p id="par0120" class="elsevierStylePara elsevierViewall"><a class="elsevierStyleCrossRef" href="#fig0010">Figure 2</a> shows the alignment of amplified <span class="elsevierStyleItalic">pks15&#47;1</span> gene sequences&#46; Strains belonging to lineage 2&#44; including the Beijing genotype&#44; maintain the pks15&#47;1 gene intact&#59; while those showing a 7-bp deletion belong to other lineages&#44; such as strains 394&#44; 123 and H37Rv&#46; Interestingly&#44; two strains identified as Beijing by spoligotyping did not show an intact <span class="elsevierStyleItalic">pks15&#47;1</span> gene&#46; The analysis of the NTF region with respect to the number of <span class="elsevierStyleItalic">IS6110</span> insertions classified the Beijing strains as modern&#44; except for strains 123 and 394&#44; which were considered pseudo-Beijing based on this result and other additional observations&#46; Most of the strains exhhibited two insertions &#40;<a class="elsevierStyleCrossRef" href="#tbl0015">Table 3</a>&#41;&#46;</p><elsevierMultimedia ident="fig0010"></elsevierMultimedia><elsevierMultimedia ident="tbl0015"></elsevierMultimedia><p id="par0125" class="elsevierStylePara elsevierViewall">All the isolates identified as Beijing strains by spoligotyping were subjected to LSPs analysis&#46; The RD 105&#44; 207 and 181 regions were deleted in all of them &#40;<a class="elsevierStyleCrossRef" href="#fig0015">Fig&#46; 3</a>&#41;&#46; Based on the subsequent deletion of RD 150 and 142&#44; the strains were grouped into three classes&#58; &#40;1&#41; strains with both RD 150 and 142 deleted &#40;1 strain&#41;&#59; &#40;2&#41; strains with RD 150 deleted and RD 142 present &#40;13 strains&#41;&#59; &#40;3&#41; strains with both RD 150 and 142 present&#46; Phylogenetically unrelated strains constitute 8&#37; of the total &#40;strains 394 and 123&#41;&#46; These results are shown in <a class="elsevierStyleCrossRef" href="#fig0015">Figure 3</a>&#46;</p><elsevierMultimedia ident="fig0015"></elsevierMultimedia></span><span id="sec0070" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0095">Discussion</span><p id="par0130" class="elsevierStylePara elsevierViewall">The <span class="elsevierStyleItalic">M&#46; tuberculosis</span> Beijing genotype is the predominant strain in many Asian countries&#46; Due to its rapid spread worldwide&#44; it is gaining significant attention&#46; Moreover&#44; its connection to drug resistance&#44; increased virulence and faster transmission rate highlights the epidemiological relevance of identifying this strain&#46; The strain was first described in the 1990s as strain &#8220;W&#8221;&#44; being the causative agent of tuberculosis outbreaks in New York&#46; The analysis revealed that the strain was a variant originating in East Asia&#46; Genetic profile studies using specific markers have shown a wide variability within the Beijing family&#44; resulting in different profiles of drug resistance and virulence&#46;<a class="elsevierStyleCrossRef" href="#bib0230"><span class="elsevierStyleSup">18</span></a></p><p id="par0135" class="elsevierStylePara elsevierViewall">The multidrug resistant <span class="elsevierStyleItalic">M&#46; tuberculosis</span> W strains that were isolated during the outbreaks in New York carried two <span class="elsevierStyleItalic">IS6110</span> sequence insertions in the NTF region&#44; and are considered modern strains&#44; unlike the common strains in Asia carrying only one <span class="elsevierStyleItalic">IS6110</span> insertion and considered ancestral strains&#46;<a class="elsevierStyleCrossRefs" href="#bib0225"><span class="elsevierStyleSup">17&#44;28</span></a> Among our isolates&#44; we found five strains with a single <span class="elsevierStyleItalic">IS6110</span> insertion and 16 strains with two <span class="elsevierStyleItalic">IS6110</span> insertions&#46; Probably&#44; the strains with two insertions were derived from those with one insertion&#46; Strain variants may emerge constantly&#44; but only some of them exhibit greater drug resistance&#44; improved virulence&#44; and transmission capabilities&#44; making them more successful to spread within the population&#46; Although Beijing strains are associated with drug resistance&#44; our results show that strains isolated in the State of Nuevo Leon are susceptible to first-line drugs&#44; with only one strain displaying resistance to isoniazid&#46;</p><p id="par0140" class="elsevierStylePara elsevierViewall">The absence of specific regions in mycobacteria is the result of unique and irreversible events producing biological markers that allow to build robust phylogenies&#46;<a class="elsevierStyleCrossRefs" href="#bib0190"><span class="elsevierStyleSup">10&#44;13&#44;25</span></a> Beijing strains from lineage 2 lack RD 105&#44; which identifies them as coming from East Asia&#46; This lineage also lacks RD207&#44; which includes spacers 1 to 34&#44; thereby giving them the distinctive spoligotyping profile&#46; Additionally&#44; some variants have lost the RD181 region&#44; and are considered modern Beijing strains&#59; among them&#44; some have also lost RD142 and RD150&#46;<a class="elsevierStyleCrossRef" href="#bib0160"><span class="elsevierStyleSup">4</span></a> The mycobacteria isolated in this work are considered modern strains as they have two <span class="elsevierStyleItalic">IS6110</span> insertions in the NTF region and lack the TbD1 region&#46; Two strains of this group had the RD181 and RD105 regions&#44; but lack the RD207 region&#46;</p><p id="par0145" class="elsevierStylePara elsevierViewall">Ninety-two percent &#40;22&#41; of the isolates belong to phylogenetic group 1&#59; four percent &#40;1&#41; to group 2 and four percent &#40;1&#41; to group 3 according to the Sreevatsan classification&#46;<a class="elsevierStyleCrossRef" href="#bib0245"><span class="elsevierStyleSup">21</span></a> We originally expected that all strains were classified in group 1 &#40;East Asia&#41;&#44; but the analysis showed that strain 123 &#40;group 2&#41; and 394 &#40;group 3&#41; have a 7-bp deletion in the <span class="elsevierStyleItalic">pks15&#47;1</span> gene&#44; which the Beijing strains do not have&#46; The <span class="elsevierStyleItalic">pks15&#47;1</span> gene is involved in the biosynthesis of phenolic glycolipids &#40;PGLs&#41; and is polymorphic in members of <span class="elsevierStyleItalic">M&#46; tuberculosis</span> complex&#46;<a class="elsevierStyleCrossRef" href="#bib0165"><span class="elsevierStyleSup">5</span></a> While Beijing strains have the <span class="elsevierStyleItalic">pks15&#47;1</span> gene intact&#44; strain H37Rv has the 7-pb deletion and belongs to group 3&#46;<a class="elsevierStyleCrossRefs" href="#bib0155"><span class="elsevierStyleSup">3&#44;5&#44;15&#44;21</span></a></p><p id="par0150" class="elsevierStylePara elsevierViewall">All these data altogether showed that the group of strains isolated in this work belongs to the Beijing family&#44; although they lack all the typical characteristics of the Beijing genotype and therefore have a different phylogenetic origin&#46; Loss of the RD207 region is due to the insertion of the <span class="elsevierStyleItalic">IS6110</span> sequence into the RD207 region that may cause the partial or complete loss of RD207&#46;<a class="elsevierStyleCrossRefs" href="#bib0175"><span class="elsevierStyleSup">7&#44;25</span></a> Different authors found that strains with spolygotyping lacking spacers 1&#8211;34 lost a bigger or smaller region than the equivalent to RD207 &#40;7399<span class="elsevierStyleHsp" style=""></span>bp&#41; and this happens in strains not belonging to group 1 of the Sreevatsan classification&#44;<a class="elsevierStyleCrossRef" href="#bib0245"><span class="elsevierStyleSup">21</span></a> which are known as pseudo-Beijing strains&#46; We observed the same in this study and the loss of spacers 1&#8211;34 may have been produced by convergent evolution as suggested&#46;<a class="elsevierStyleCrossRef" href="#bib0175"><span class="elsevierStyleSup">7</span></a> The latter study included pseudo-Beijing strains within lineage 3&#59; however&#44; we found that our strains belong to lineage 4&#44; suggesting a higher prevalence of group 4 &#40;Euro-American&#41; in this geographic region&#46;</p><p id="par0155" class="elsevierStylePara elsevierViewall">In order to understand the phylogenetic relationship among the isolates&#44; the number of clusters and the unique genotypes a minimum spanning tree &#40;MST&#41; was constructed using the Bionumerics v7 software &#40;Applied Maths NV&#44; Sint-Martens-Latem&#44; Belgium&#41;&#46; According to this analysis&#44; it is highly likely that the infection of patients with unique strains was the result of sporadic contact&#44; reactivation of that sporadic contact or imported cases&#46; Moreover&#44; infections caused by strains grouped into clusters exhibit a clonal relationship&#44; although we could not establish a direct transmission&#44; as they were collected in different years&#46; Probably&#44; this genotype has existed in the region for several decades&#44; becoming an endemic genotype isolated sporadically&#46; Additionally&#44; most isolates are sensitive to first-line drugs&#46;<a class="elsevierStyleCrossRefs" href="#bib0270"><span class="elsevierStyleSup">26&#44;22</span></a></p><p id="par0160" class="elsevierStylePara elsevierViewall">The MIRU-VNTR analysis showed that the MIRUs with higher allelic diversity and therefore better discriminatory power to distinguish Beijing strain sublineages were MIRU10&#44; MIRU31&#44; MIRU39&#44; MRU40&#44; and MIRU 26&#46; The variability found with these markers suggests that the isolates did not have the same origin&#46; The dendrogram based on the 18 MLVAs reveals that strains 123 and 394 do not have any relationship with Beijing strains and that the isolates from the State of Nuevo Leon and those reported from Baja California belong to two different populations&#46; Strains 110 and 130 did not belong to any of these groups and&#44; therefore&#44; they might be imported or reactivation cases&#46; The State of Baja California has a high commercial activity with Asian countries&#46; Tijuana is one of the main seaports in Mexico and during the past year&#44; thousands of Central American immigrants have arrived in the city&#46; Mexicali&#44; located 180<span class="elsevierStyleHsp" style=""></span>km away from Tijuana&#44; houses the largest Chinese community in Mexico&#46; Trade and human immigration from these areas might have facilitated the introduction of the Asian lineage and its later dissemination to the rest of the country&#44; which could have resulted in the generation of sublineages as was observed in this study&#46; Records from the international spoligotyping database up to 2006 showed than Beijing and Beijing-like strains represent around 50&#37; of the strains in Eastern Asia and 13&#37; of the isolates worldwide&#46;<a class="elsevierStyleCrossRef" href="#bib0235"><span class="elsevierStyleSup">19</span></a> The percentage of the Beijing genotype in previous studies had been close to 1&#46;3&#37;&#46; However&#44; among the 406 strains analyzed in this study&#44; the percentage was 3&#46;4&#37;&#44; implying a dissemination of the genotype&#44; yet this prevalence has not manifested itself as an outbreak&#46;</p></span><span id="sec0075" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0100">Funding</span><p id="par0165" class="elsevierStylePara elsevierViewall">The authors acknowledge project 568475 of the Frontier Sciences Fund-CONACYT for supporting this research&#44; and University of Monterrey for financing the publication fees&#46;</p></span><span id="sec0080" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0105">Authors&#8217; contributions</span><p id="par0170" class="elsevierStylePara elsevierViewall">PBM and FGS &#8211; funding acquisition&#59; NCB&#44; JCG&#44; HAR&#44; MBM&#44; AMV&#44; VVM &#8211; methodology&#59; NCB&#44; PBM and VVM &#8211; data curation&#59; NCB&#44; PBM and VVM &#8211; formal analysis&#59; JCG&#44; HAR&#44; MBM and AMV &#8211; supervision&#59; NCB&#44; PBM&#44; FGS&#44; HAR and VVM &#8211; manuscript writing&#59; review and editing &#8211; all authors&#46;</p></span><span id="sec0085" class="elsevierStyleSection elsevierViewall"><span class="elsevierStyleSectionTitle" id="sect0110">Conflict of interests</span><p id="par0175" class="elsevierStylePara elsevierViewall">The author&#40;s&#41; declared no potential conflicts of interest with respect to the research&#44; authorship&#44; and&#47;or publication of this article&#46;</p></span></span>"
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    "highlights" => array:2 [
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      "resumen" => "<span id="abst0005" class="elsevierStyleSection elsevierViewall"><p id="spar0005" class="elsevierStyleSimplePara elsevierViewall"><ul class="elsevierStyleList" id="lis0005"><li class="elsevierStyleListItem" id="lsti0005"><span class="elsevierStyleLabel">&#8226;</span><p id="par0005" class="elsevierStylePara elsevierViewall">We determine the genotypes MTB belonging to the Beijing lineage&#46;</p></li><li class="elsevierStyleListItem" id="lsti0010"><span class="elsevierStyleLabel">&#8226;</span><p id="par0010" class="elsevierStylePara elsevierViewall">406 MTB isolates were genotyped with Spoligotyping and 18-locus MIRU-VNTR&#46;</p></li><li class="elsevierStyleListItem" id="lsti0015"><span class="elsevierStyleLabel">&#8226;</span><p id="par0015" class="elsevierStylePara elsevierViewall">Beijing genotype characterized phylogenetically&#46;</p></li><li class="elsevierStyleListItem" id="lsti0020"><span class="elsevierStyleLabel">&#8226;</span><p id="par0020" class="elsevierStylePara elsevierViewall">Genomic diversity of MTB in Monterrey&#44; Mexico&#46;</p></li></ul></p></span>"
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        "resumen" => "<span id="abst0010" class="elsevierStyleSection elsevierViewall"><p id="spar0010" class="elsevierStyleSimplePara elsevierViewall">Tuberculosis remains a serious threat to human health as an infectious disease in Mexico&#46; Data about the genotypes of circulating <span class="elsevierStyleItalic">Mycobacterium tuberculosis</span> isolates &#40;MTB&#41; in the State of Nuevo Leon&#44; Mexico are scarce&#46; We aimed to determine the genotypes of circulating MTB belonging to the Beijing lineage recovered from patients in the State of Nuevo Leon&#44; Mexico&#46; A total of 406 MTB isolates from this state were genotyped using the spoligotyping method and 18-locus MIRU-VNTR&#46; Lineage classification and MTB transmission analysis were performed&#46; Based on the spoligotyping analysis&#44; we found 24 strains belonging to the Beijing genotype that were characterized phylogenetically&#46; The MIRUs showed greater discriminatory power than the standard RFLP-IS6110 method&#59; therefore&#44; the greatest allelic diversity among the Beijing strains was observed with MIRU10&#44; MIRU31&#44; MIRU39&#44; MRU40&#44; and MIRU 26&#46; MVLA analysis showed a profile variation between Beijing and non-Beijing strains&#46; The minimum spanning tree &#40;MST&#41; showed that 79&#37; &#40;19&#41; of the strains are related&#46; All Beijing strains exhibited the deletion of region TbD1&#44; which is a characteristic of modern strains&#46; The application of spoligotyping and MIRU-VNTR-18 methods together proved to be more sensitive&#44; discriminatory&#44; and rapid than the standard method for the epidemiological analysis of <span class="elsevierStyleItalic">Mycobacterium Beijing</span> isolates&#46; This study is one of the first to describe the genomic diversity of <span class="elsevierStyleItalic">M&#46; Beijing</span> in the State of Nuevo Leon&#44; Mexico&#46;</p></span>"
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        "titulo" => "Resumen"
        "resumen" => "<span id="abst0015" class="elsevierStyleSection elsevierViewall"><p id="spar0015" class="elsevierStyleSimplePara elsevierViewall">La tuberculosis sigue siendo una grave amenaza para la salud humana como enfermedad infecciosa en M&#233;xico&#46; Los datos sobre los genotipos de los aislados de <span class="elsevierStyleItalic">Mycobacterium tuberculosis</span> &#40;MTB&#41; circulantes en el estado mexicano de Nuevo Le&#243;n son escasos&#46; Nuestro objetivo fue determinar los genotipos circulantes de MTB pertenecientes al linaje Beijing recuperados de pacientes en ese estado&#46; Un total de 406 aislamientos de MTB del estado de Nuevo Le&#243;n&#44; M&#233;xico&#44; fueron genotipados con el m&#233;todo espoligotipado y MIRU-VNTR de 18 locus&#46; Se realizaron clasificaci&#243;n de linaje y an&#225;lisis de transmisi&#243;n de MTB&#46; Con base en el an&#225;lisis de espoligotipado&#44; encontramos 24 cepas pertenecientes al genotipo Beijing y se caracterizaron filogen&#233;ticamente&#46; Los MIRU mostraron un mayor poder discriminatorio que el m&#233;todo est&#225;ndar de RFLP-IS6110&#44; mayor diversidad al&#233;lica entre las cepas de Beijing se observ&#243; con MIRU10&#44; MIRU31&#44; MIRU39&#44; MRU40 y MIRU 26&#46; El an&#225;lisis de MVLA mostr&#243; una variaci&#243;n de perfil entre las cepas de Beijing y no Beijing&#46; El &#225;rbol de expansi&#243;n m&#237;nima &#40;MST&#41; mostr&#243; que el 79&#37; &#40;19&#41; de las cepas est&#225;n relacionadas&#46; Todas las cepas de Beijing ten&#237;an la deleci&#243;n de la regi&#243;n TbD1&#44; caracter&#237;stica de las cepas modernas&#46; La aplicaci&#243;n de los m&#233;todos de espoligotipado y MIRU-VNTR-18 en conjunto result&#243; ser m&#225;s sensible&#44; discriminatoria y r&#225;pida que el m&#233;todo est&#225;ndar para el an&#225;lisis epidemiol&#243;gico de aislamientos de <span class="elsevierStyleItalic">Mycobacterium</span> Beijing&#46; Este estudio es uno de los primeros en describir la diversidad gen&#243;mica de <span class="elsevierStyleItalic">Mycobacterium</span> Beijing en el Estado mejicano de Nuevo Le&#243;n&#46;</p></span>"
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    ]
    "apendice" => array:1 [
      0 => array:1 [
        "seccion" => array:1 [
          0 => array:4 [
            "apendice" => "<p id="par0190" class="elsevierStylePara elsevierViewall">The followings are the supplementary data to this article&#58;<elsevierMultimedia ident="upi0005"></elsevierMultimedia></p>"
            "etiqueta" => "Appendix B"
            "titulo" => "Supplementary data"
            "identificador" => "sec0095"
          ]
        ]
      ]
    ]
    "multimedia" => array:64 [
      0 => array:7 [
        "identificador" => "fig0005"
        "etiqueta" => "Figure 1"
        "tipo" => "MULTIMEDIAFIGURA"
        "mostrarFloat" => true
        "mostrarDisplay" => false
        "figura" => array:1 [
          0 => array:4 [
            "imagen" => "gr1.jpeg"
            "Alto" => 1602
            "Ancho" => 2050
            "Tamanyo" => 116922
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        ]
        "descripcion" => array:1 [
          "en" => "<p id="spar0020" class="elsevierStyleSimplePara elsevierViewall">The minimum spanning tree &#40;MST&#41; showed than 79&#37; &#40;19&#41; of the strains are related&#46;</p>"
        ]
      ]
      1 => array:7 [
        "identificador" => "fig0010"
        "etiqueta" => "Figure 2"
        "tipo" => "MULTIMEDIAFIGURA"
        "mostrarFloat" => true
        "mostrarDisplay" => false
        "figura" => array:1 [
          0 => array:4 [
            "imagen" => "gr2.jpeg"
            "Alto" => 706
            "Ancho" => 1275
            "Tamanyo" => 234953
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        "descripcion" => array:1 [
          "en" => "<p id="spar0025" class="elsevierStyleSimplePara elsevierViewall">It shows the alignment of amplified <span class="elsevierStyleItalic">pks15&#47;1</span> gene sequences&#46; Strains of lineage 2&#44; where the genotype Beijing is included&#44; keep the pks15&#47;1 gene intact&#59; while those showing a 7p-deletion belong to other lineages&#44; such as strains 394&#44; 123 and H37Rv&#46;</p>"
        ]
      ]
      2 => array:7 [
        "identificador" => "fig0015"
        "etiqueta" => "Figure 3"
        "tipo" => "MULTIMEDIAFIGURA"
        "mostrarFloat" => true
        "mostrarDisplay" => false
        "figura" => array:1 [
          0 => array:4 [
            "imagen" => "gr3.jpeg"
            "Alto" => 972
            "Ancho" => 1350
            "Tamanyo" => 122526
          ]
        ]
        "descripcion" => array:1 [
          "en" => "<p id="spar0030" class="elsevierStyleSimplePara elsevierViewall">The strains analyzed in this study came from a common ancestor of <span class="elsevierStyleItalic">M&#46; tuberculosis</span>&#44; which branched into lineage 2 from Eastern Asia &#40;in blue&#41; and lineage 4 Euro-American &#40;in red&#41;&#46; Lineage 2 is represented by a continuous blue line emerging from the root and with the deletions RD105&#44; RD207 and RD181&#59; this line divides into 3 clades&#44; the first one has the RDs 150 and 142&#44; the second clade lacks RD 150 but has RD 142&#44; while the third clade lacks both RDs 150 and 142&#46; Lineage 4&#44; represented by a red line emerging from the root&#44; has RDs 105&#44; 207&#44; 181&#44; 150 and 142&#46;</p>"
        ]
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      3 => array:8 [
        "identificador" => "tbl0005"
        "etiqueta" => "Table 1"
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        "detalles" => array:1 [
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        "tabla" => array:2 [
          "leyenda" => "<p id="spar0040" class="elsevierStyleSimplePara elsevierViewall">SIT&#58; poligotype international types&#59; &#9633;&#58; absence of spacer&#59; &#9632;&#58; presence of spacer&#59; O&#58; orphan&#46;</p>"
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                  """
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        "descripcion" => array:1 [
          "en" => "<p id="spar0035" class="elsevierStyleSimplePara elsevierViewall">Spoligotypes shared by <span class="elsevierStyleItalic">Mycobacterium tuberculosis</span> strains evaluated in this study&#46;</p>"
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      4 => array:8 [
        "identificador" => "tbl0010"
        "etiqueta" => "Table 2"
        "tipo" => "MULTIMEDIATABLA"
        "mostrarFloat" => true
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        "detalles" => array:1 [
          0 => array:3 [
            "identificador" => "at2"
            "detalle" => "Table "
            "rol" => "short"
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        "tabla" => array:2 [
          "leyenda" => "<p id="spar0050" class="elsevierStyleSimplePara elsevierViewall">MIRU-VNTR&#58; mycobacterial interspersed repetitive unit variable number tandem repeat&#59; MLVA&#58; multiple loci VNTR analysis&#46;</p>"
          "tablatextoimagen" => array:1 [
            0 => array:2 [
              "tabla" => array:1 [
                0 => """
                  <table border="0" frame="\n
                  \t\t\t\t\tvoid\n
                  \t\t\t\t" class=""><thead title="thead"><tr title="table-row"><th class="td" title="\n
                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " colspan="13" align="center" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">MIRU 12</th><th class="td" title="\n
                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " colspan="6" align="center" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">MVLA &#40;Additional loci&#41;</th></tr><tr title="table-row"><th class="td" title="\n
                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">Strain&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t\t\t</th><th class="td" title="\n
                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="center" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">MIRU 02&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t\t\t</th><th class="td" title="\n
                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="center" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">MIRU 04&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t\t\t</th><th class="td" title="\n
                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="center" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">MIRU 40&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t\t\t</th><th class="td" title="\n
                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="center" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">MIRU 10&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t\t\t</th><th class="td" title="\n
                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="center" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">MIRU 16&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t\t\t</th><th class="td" title="\n
                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="center" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">MIRU 20&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t\t\t</th><th class="td" title="\n
                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="center" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">MIRU 23&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t\t\t</th><th class="td" title="\n
                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="center" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">MIRU 24&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t\t\t</th><th class="td" title="\n
                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="center" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">MIRU 26&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t\t\t</th><th class="td" title="\n
                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="center" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">MIRU 27&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t\t\t</th><th class="td" title="\n
                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="center" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">MIRU 31&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t\t\t</th><th class="td" title="\n
                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="center" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">MIRU 39&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t\t\t</th><th class="td" title="\n
                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="center" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">Mtub 21&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t\t\t</th><th class="td" title="\n
                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="center" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">QUB 11b&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t\t\t</th><th class="td" title="\n
                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="center" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">QUB 18&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t\t\t</th><th class="td" title="\n
                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="center" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">QUB 26&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t\t\t</th><th class="td" title="\n
                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="center" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">QUB 4156&nbsp;\t\t\t\t\t\t\n
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                  \t\t\t\t\ttable-head\n
                  \t\t\t\t  " align="center" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t" scope="col" style="border-bottom: 2px solid black">ETR-F&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t\t\t</th></tr></thead><tbody title="tbody"><tr title="table-row"><td class="td-with-role" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t ; entry_with_role_rowhead " align="left" valign="\n
                  \t\t\t\t\ttop\n
                  \t\t\t\t">349&nbsp;\t\t\t\t\t\t\n
                  \t\t\t\t</td><td class="td" title="\n
                  \t\t\t\t\ttable-entry\n
                  \t\t\t\t  " align="char" valign="\n
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        "texto" => "<p id="par0180" class="elsevierStylePara elsevierViewall">The authors would like to express their gratitude to the personnel of Health of the State of Nuevo Leon&#44; Mexico for support and collaboration in this study&#46;</p>"
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