
 target population - the target population consisted of 120 tb patients 
 living in the mam who were ensured by the mexican institute of social security ( imss 2014 ) .
the imss provides health care for 83.5% 
 of the patients in nuevo len . during a 16-month period ,
all patients who visited the 
 units of familiar medicine of the mam with symptoms suggestive of ptb [ 268 ( 100% of the 
 suspicious cases in the mam in the care of the imss ) ] were included in this study . a 
 total of 134 patients had mtb - positive sputum samples and 120 of these patients agreed 
 to participate in this study .
the study group was composed of 64% men and 36% women 
 between the ages of 13 - 84 years ( median = 49 years ) .
the range of ages was 18 - 68 years 
 and the time of residence in the mam ranged from less than one year to 68 years ( mean  
 standard deviation = 34.06  16.03 years ) .
the participants presented clear symptoms of 
 ptb ( who 2014 ) and acid - fast bacilli ( afb ) in their sputum samples . ptb patients 
 attended their respective family medicine clinics or their zone hospitals during the 
 study . in these facilities ,
afb were detected and reported by a chemist working in the clinical 
 laboratory of each medical facility . 

collection of data regarding potential tb risk factors - the collection 
 of epidemiological data and the isolation , mtb typing and genotyping were performed in 
 the northeast biomedical research centre ( cibin ) of the imss .
the patients with a 
 clinical diagnosis of ptb signed an informed consent form and answered a questionnaire 
 to provide appropriate information regarding the factors that could favour tb 
 transmission ( such as crowded places frequented by the ptb patients ) .
familial contacts 
 and human immunodeficiency virus / acquired immune deficiency syndrome status were sought 
 and correlated with the clusters ; additionally , groups of clusters were analysed , but 
 because the data were negative , these results are not presented in this article . 

social networks and risk factors - the elements of social networks 
 analysed in this work were family medical clinics , zonal general hospitals , 
 municipalities and familial contacts .
the risk factors examined were comorbidities , 
 patterns of resistance or sensitivity to first line anti - tubercular drugs , occupation 
 and drug consumption ( alcoholism , tobacco use and psychotropic substance abuse ) . 

control mtb strain - the mtb h37rv strain ( american type culture 
 collection , catalogue 27294 ) was included as an internal genotyping control . 

mtb isolates - each participant donated up to three sputum samples from 
 which mycobacteria were isolated and typified . immediately after collection ,
the samples 
 were transported to our laboratory in the cibin , decontaminated and the bacteria were 
 concentrated by centrifugation .
a smear from every sputum sample was prepared and 
 stained using the ziehl - neelsen method and afb were microscopically examined ( ellis & zabrowarny 1993 ) .
each of these samples 
 was inoculated onto lowenstein - jensen ( lj ) slants ( becton dickinson , usa ) and incubated 
 in a 5% co2 atmosphere at 37c for two - five weeks .
slants with typical mtb 
 colonies were typified ( kent & kubica 1985 , 
 balandrano - campos et al . 1996 ) and stored at 
 -70c until use .
recovery of frozen isolates - the frozen mtb isolates were thawed at 
 -20c overnight and then at 4c for 3 - 4 h. mycobacteria were inoculated on lj slants and 
 incubated at 37c until colonies were clearly visible . 

drug sensitivity tests for first - line anti - mtb drugs - the tests were 
 performed with a bactec 460 radiometric system ( siddiqi 1996 ) .
the assayed drugs were 
 isoniazid ( inh ) ( 0.1 g / ml ) , streptomycin ( 2.0 g / ml ) , rifampicin ( rif ) ( 2.0 g / ml ) , 
 ethambutol ( 2.5 g / ml ) and pyrazinamide ( 100 mg / ml ) .
extraction of mtb dna - under aseptic conditions , two loops of 
 mycobacteria colonies were transferred to a glass test tube containing 2.5 ml of 
 tris - edta buffer [ 10 mm tris / hcl ph 8) ( amresco , usa ) and 1.0 mm edta ( promega , usa ) ] 
 and five - six glass beads . the suspension was homogenised using a vortex and a 0.5-ml 
 aliquot was transferred to a 1.5-ml capped conical polypropylene tube ( eppendorf , 
 germany ) and inactivated in an 80c water bath ( terlab m40 , mexico ) for 50 min .
dna was 
 extracted from mycobacteria according to the ctab ( hexadecyltrimethyl - ammonium bromide ) 
 method ( van soolingen et al . 2001 ) . 

( 1997 ) using a commercial kit 
 ( isogen life science , the netherlands ) .
( 1993 ) 
 and miru - vntr analyses were performed according to cowan 
 et al .
, the following 15 loci were analysed : 
 miru 4 , 10 , 16 , 26 , 31 and 40 ( cowan et al .
2002 ) 
 as well as mtub 04 , etr c , etr a , qub 11b , mtub21 , qub26 , mtub30 , mtub39 and qub4156 
 ( supply et al .
( 2013 ) , we defined clusters as 
 those ptb cases with indistinguishable mtb genotypes .
the spoligotypes were converted to 
 their corresponding octal codes as described by dale et 
 al .
( 2001 ) and clusters and unique patterns were identified using spss 
 software , v.10.0 .
the rflp - is6110 data were analysed with bionumerics 
 software , v.2.0 ( applied maths , belgium ) using the unweighted pair group method with an 
 arithmetical mean and dice s coefficient .
the running differences in the gels were 
 normalised using the relative position of the /hind iii and x174/hae iii molecular 
 weight markers ( sigma - aldrich ) using bionumerics software . to determine the best 
 tolerance ,
when 
 the alignments of all of the reference rflp - is6110 patterns from all 
 the agarose gels matched , the tolerance value was set at 1.9% .
the genotypes were 
 considered identical according to the results from the analyses performed by the 
 bionumerics program .
because rflp - is6110 codes have not been previously 
 described , we designed a code using the relative migration of each is6110 
 band ( rf ) of every mtb isolate dna sample ( fig .
1 ) . the description of the rflp - is6110 codes 
 is as follows : the rflp - is6110 codes have two arms of numbers , 
 separated by a dot .
the left arm is composed of one or two digits and includes 
 information regarding the number of bands found in each mtb isolate .
the right arm 
 contains the rf of each dna band with an is-6110 element . 

each rf is represented by three numbers . from left to right , the 
 rf values were placed in descending order of dna - molecular weight .
the 
 rf values were calculated by dividing the distance between the origin and 
 front of the electrophoresis by the distance travelled by each 
 is6110-band and multiplying by 1  10 .
the miru - vntr 
 patterns were analysed with microsoft excel 2002 software ( microsoft co , usa ) .
the cluster groups with comparable codes were named as follows to differentiate the 
 clusters from the genotyping methods : spoligotypes , miru - vntr and 
 rflp - is6110 , smr , spoligotypes and rflp - is6110 , sr , 
 spoligotypes and miru - vntr , sm , miru - vntr and rflp - is6110 , mr , 
 spoligotypes , s , rflp - is6110 , r , and miru - vntr , m. the methods are 
 referred to by their corresponding shortened names ( spoligotyping , miru - vntr and 
 rflp - is6110 ) . 

these were generated by the bionumerics software and 
 show the relationships of clusters and groups of clusters using the 
 rflp - is6110 method exclusively .
venn diagram - the venn diagram describes the metrics by which the 120 
 mtb isolates were organised using spoligotyping , miru - vntr and 
 rflp - is6110 .
the diagram was constructed using powerpoint software 
 for windows 7 ( microsoft co ) with the data depicted in table i. 
 statistics - the significance ( p = 0.05 ) for correlation between the 
 aforementioned cluster - groups and the ptb characteristics was calculated by spearman s 

. statistical correlation referred to the relationships that involved the dependence of 
 code - set belonging to each mtb cluster group having comparable genetic codes and each 
 ptb patient characteristic . 

ethics - this protocol represented a minor risk for tb patients and was 
 authorised by the scientific and ethical committee of the cibin / imss ( protocol 
 r-2005 - 1908 - 2 ) .
1 shows the rflp - is6110 
 patterns and dendrogram of the 120 ptb clinical isolates .
2 ) describes the metrics by which the 120 mtb 
 isolates were organised using spoligotyping , miru - vntr and rflp - is6110 . 

while 70.83% ( 85 ) of the isolates were clustered , 29.17% ( 35 ) of the isolates ( unique or 
 orphan cases ) were not clustered by any genotyping method .
the clusters constituted the 
 following groups with respect to the 120 mtb isolates : smr , 5% ( 6 isolates ) , sm , 12.50% 
 ( 15 isolates ) , sr , 1.60% ( 2 isolates ) , m , 5% ( 6 isolates ) and s , 46.67% ( 56 isolates ) . 

supplementary data , table i shows each case identified by the number assigned by our 
 laboratory and its corresponding s , m or r code .
supplementary data , table ii retains the identical organisation of the 
 mtb cases as clusters and groups of clusters , as in supplementary data , table i. supplementary data , table ii depicts the factors that could favour ptb transmission in 
 our target population .
five groups were formed with comparable codes for spoligotyping , 
 miru - vntr and rflp - is6110 ( smr ) , a combination of two codes ( sr and sm ) 
 or only one ( s and m ) .
group six was formed by orphan cases . additionally , supplementary 
 data , table ii shows that the variables with the 
 highest frequencies were first ( family medicine clinic 35 ) and second level medical 
 facilities ( zonal general hospitals 4 and 17 ) , municipalities ( guadalupe and monterrey ) , 
 comorbidities ( diabetes mellitus type 2 ) , occupation ( car painting , mining and 
 industrial production ) , multidrug resistance ( to at least rif and inh ) and drug 
 consumption ( tobacco use and alcoholism ) . 

schematic representation by which the 120 
 mycobacterium tuberculosis - isolates included in this study 
 were organised by spoligotyping , mycobacterial interspersed repetitive 
 units - variable number tandem repeats ( miru - vntr ) and the conventional 
 restriction fragment length polymorphism ( rflp)-is6110 
 analysis .
association of cluster groups with situations favouring tb transmission 
 - tables i , ii show the association of cluster groups with crowded places frequented by 
 ptb patients and risk factors .
smr presented a significant correlation with family 
 medicine clinics , municipalities , comorbidities and drug consumption . sr and factors 
 favouring mtb transmission were not calculated because the number of cases was 
 insufficient .
no relationship was found 
 between a cluster group and members of the same family or neighbours .
we showed that a combination of rflp - is6110 , miru - vntr and 
 spoligotyping is essential to identify mtb clusters , to group the clusters and to 
 discard orphan cases . using these data
, we investigated how social networks and risk 
 factors correlated with the cluster groups ( glynn et al . 

smr was the only group that 
 correlated with two of the three elements of social networks and with two of the four 
 risk factors considered in this study .
our data strongly suggest that when tb surveys 
 are performed in a metropolis , such as the mam , the more markers that are considered in 
 performing a correlation analysis with risk factors , the more reliable the connection 
 with a ptb epidemic focus would be .
the factors that correlated with smr substantiated a recent mtb transmission . regarding 
 social networks ( family medicine clinics , primary clinics and municipalities ) ,
clinic waiting rooms are closed 
 environments with recirculated air , in which bacillipherous patients are seated by 
 individuals without ptb . during standard treatment , patients repeat this type of visit 
 several times . no correlation was observed between smr and zonal general hospitals , 
 possibly because tb patients are treated in family medicine clinics by an epidemiologist 
 until the case of tb is cured or the patient is referred to a pulmonologist at a 
 specialty hospital .
thus , the risk of ptb transmission in second level facilities is 
 markedly reduced .
ptb transmission throughout social networks has been previously 
 reported ; garca - garca et al .
( 2000 ) found an rflp - is6110 cluster that 
 developed after ptb micro - epidemics occurred in a social network formed by assiduous 
 clients attending clandestine bars .
in addition to first level medical facilities , 
 workers and housewives typically frequent the same crowded public places , such as 
 schools , markets , stores , cinemas , churches or bars and are transported by urban buses . 

these factors are 
 recognised as risk factors ( davies 2005 ) and we 
 identified the correlations of diabetes and drug abuse with smr .
the application of a combination of spoligotyping , miru - vntr and 
 rflp - is6110 and an analysis of spearman s correlation of these 
 genotypes with factors that could facilitate mtb transmission is required to discover 
 new ptb micro - epidemics that are possible in a metropolitan population .
considering the 
 increase in multidrug resistant and extended drug resistant mtb strains that threaten tb 
 control worldwide ( who 2014 ) , the recent 
 development of strains resistant to all first and second line anti - tb drugs , which have 
 been identified as completely mtb resistant ( totally drug - resistant - mtb ) ( rowland 2012 ) , could be useful for applying analyses 
 such as this one to describe future tb surveys or potential epidemic foci of ptb . 

table iassociation of cluster - groups and social networksgroup 
 level of medical 
 attention 
 clinics of familiar 
 medicinezonal general 
 hospitalsmunicipality 

 spearman s 
 correlationsmr 
 0.033 
 0.1660.013srnc 
 ncncsm0.6770.2560.380s0.0780.1050.316m0.7790.5590.261a : groups of clusters having comparable codes;b : not computable ( nc ) because these groups were constituted just by one 
 cluster having two cases .
capitals correspond to groups of clusters showing 
 comparable codes of spoligotyping ( s ) , mycobacterial interspersed repetitive 
 units - variable number tandem repeats ( m ) or restriction fragment length 
 polymorphism - is6110 ( r ) .
groups of clusters having comparable codes ; : not computable ( nc ) because these groups were constituted just by one 
 cluster having two cases .
capitals correspond to groups of clusters showing 
 comparable codes of spoligotyping ( s ) , mycobacterial interspersed repetitive 
 units - variable number tandem repeats
table iiassociation of cluster - groups with risk factorsgroupcomorbiditypattern of drug 
 resistanceoccupationdrug consumption 

 spearman s correlationsmr 
 0.047 
 0.6431.000 
 0.012 
 srnc 
 ncncncsm0.3810.2780.2340.531s0.1960.4000.0670.271
m 
 0.047 
 0.4921.0000.391a : not computable ( nc ) because these groups were constituted just by one 
 cluster having two cases .
capitals correspond to groups of clusters showing 
 comparable codes of spoligotyping ( s ) , mycobacterial interspersed repetitive 
 units - variable number tandem repeats
not computable ( nc ) because these groups were constituted just by one 
 cluster having two cases .
capitals correspond to groups of clusters showing 
 comparable codes of spoligotyping ( s ) , mycobacterial interspersed repetitive 
 units - variable number tandem repeats ( m ) or restriction fragment length 
 polymorphism - is6110 ( r ) .
 the characteristics of tuberculosis ( tb ) patients related to a chain of recent tb 
 transmissions were investigated . 
 mycobacterium tuberculosis ( mtb ) isolates ( 120 ) were 
 genotyped using the restriction fragment length polymorphism - is6110 ( r ) , spacer 
 oligotyping ( s ) and mycobacterial interspersed repetitive units - variable number of 
 tandem repeats ( m ) methods . 
 the mtb isolates were clustered and the clusters were 
 grouped according to the similarities of their genotypes . 
 spearman s rank correlation 
 coefficients between the groups of mtb isolates with similar genotypes and those 
 patient characteristics indicating a risk for a pulmonary tb ( ptb ) chain transmission 
 were ana- lysed . 
 the isolates showing similar genotypes were distributed as follows : 
 smr ( 5% ) , sm ( 12.5% ) , sr ( 1.67% ) , mr ( 0% ) , s ( 46.67% ) , m ( 5% ) and r ( 0% ) . 
 the 
 remaining 35 cases were orphans . 
 smr exhibited a significant correlation ( p < 
 0.05 ) with visits to clinics , municipalities and comorbidities ( primarily diabetes 
 mellitus ) . 
 s correlated with drug consumption and m with comorbidities . 
 smr is needed 
 to identify a social network in metropolitan areas for ptb transmission and s and m 
 are able to detect risk factors as secondary components of a transmission chain of 
 tb . 
PATIENTS, MATERIALS AND METHODS
RESULTS
DISCUSSION