Mycobacterium tuberculosis population structure and molecular … · 2018. 3. 21. · Boleíta 1,...

Mycobacterium tuberculosis populationstructure and molecular epidemiologicalanalysis in Sucre municipality,Miranda state, Venezuela.

Margareth A. Patiño1, Edgar Abadía1. Solalba Gómez2, Mailis Maes3, Mariana Muñoz1,

Daniela Gómez1, Patricia Guzmán1, María Victoria Méndez1, Carmen Ramirez4,

Mercedes España4, Jacobus de Waard3 and Howard Takiff 1

1Laboratorio de Genética Molecular, Instituto Venezolano de InvestigacionesCientíficas. Altos de Pipe. Venezuela.

2Laboratorio “Luis Quiñones”. TB Sucre, Distrito #7 de Salud, Edo. Miranda, Venezuela.Laboratorio de Tuberculosis del Instituto de Biomedicina. Caracas, Venezuela.

3Laboratorio de Tuberculosis del Instituto de Biomedicina. Caracas, Venezuela.4Instituto de Tuberculosis. Algodonal, Caracas, Venezuela.

Abstract. Sucre municipality is a large, densely populated marginal areain the eastern part of Caracas, Venezuela that consistently has more cases oftuberculosis than other municipalities in the country. To identify the neigh-borhoods in the municipality with the highest prevalence of tuberculosis, anddetermine whether the Mycobacterium tuberculosis strain distribution in thismunicipality is different from that previously found in the western part of Ca-racas and the rest of Venezuela, we collected data on all tuberculosis cases inthe municipality diagnosed in 2005-6. We performed two separate molecularepidemiological studies, spoligotyping 44 strains in a first study, andspoligotyping 131 strains, followed by MIRU-VNTR 15 on 21 clustered isolatesin the second. With spoligotyping, the most common patterns were Shared In-ternational Type SIT17 (21%); SIT42 (15%); SIT93 (11%); SIT20 (7%); SIT53(6%), a distribution similar to other parts of Venezuela, except that SIT42 andSIT20 were more common. MIRU-VNTR 15 showed that six of seven SIT17strains examined belonged to a large cluster previously found circulating inVenezuela, but all of the SIT42 strains were related to a cluster centered inthe neighborhoods of Unión and Maca, with a MIRU-VNTR pattern not previ-ously seen in Venezuela. It appears that a large percentage of the tuberculosisin the Sucre municipality is caused by the active transmission of two strainfamilies centered within distinct neighborhoods, one reflecting communica-tion with the rest of the country, and the other suggesting the insular, iso-lated nature of some sectors.

Invest Clin 55(4): 332 - 351, 2014

Corresponding author: Howard Takiff. Laboratorio de Genética Molecular, CMBC, IVIC. Km 11. Carr. Paname-ricana, Caracas 1020 A, Venezuela. E-mail: htakiff@gmail.com

Estructura poblacional y análisis epidemiológico molecular deMycobacterium tuberculosis en el municipio Sucre, estadoMiranda, Venezuela.Invest Clin 2014: 55(4): 332 - 351

Palabras clave: epidemiología molecular, genotipo, spoligotyping, Mycobacterium

tuberculosis, MIRU-VNTR, epidemiología, tuberculosis.

Resumen. El municipio Sucre es un área densamente poblada del este deCaracas, Venezuela, con más casos de tuberculosis que otros municipios delpaís. Para establecer las áreas en el municipio Sucre con la mas alta prevalen-cia de tuberculosis y determinar sí la distribución de cepas de Mycobacterium

tuberculosis es diferente de las encontradas previamente en el Oeste de Cara-cas y el resto de Venezuela, se recolectaron los datos de todos los casos diag-nosticados de tuberculosis en el municipio en el 2005-6. Además, se aplicarondos estudios de epidemiología molecular, el primero con 44 aislados en 2006y el segundo con 131 aislados del 2006 al 2011, todos caracterizados por spo-

ligotyping. Fue aplicada la técnica MIRU VNTR15 sobre 21 aislados agrupados.Con spoligotyping, los patrones encontrados fueron SIT17 (21%); SIT42(15%); SIT93 (11%); SIT20 (7%); SIT53 (6%), presentando una distribución si-milar en otras partes de Venezuela, con la diferencia de que el SIT42 y elSIT20 fueron comunes en el municipio. MIRU VNTR15 mostró que seis de lassiete cepas SIT17 pertenecían a un gran grupo encontrado previamente enVenezuela, mientras las cepas SIT42, estaban relacionados a un grupo concen-trado en los Barrios Unión y Maca, con un patrón MIRU VNTR no visto previa-mente en Venezuela. Los resultados indicarían que un gran porcentaje de tu-berculosis en el municipio Sucre es causada por transmisión activa de dos fa-milias, una reflejando comunicación con el resto del país, y otra sugiriendoque es un aislado propio de algunos Barrios del municipio.

Recibido: 8-1-2014 Aceptado: 19-6-2014

INTRODUCTION

Despite the availability of effectiveanti-tuberculosis chemotherapy for over 50years, Tuberculosis (TB) remains a majorglobal health problem. Each year there arenearly 9 million cases and an estimated 1.7million deaths worldwide, and the numberof new cases is higher than at any othertime in history (1). Fortunately, the inci-dence of TB in Venezuela is only moderate,with about 33 cases per 100.000 of inhabit-ants per year (2) but marginal and indige-

nous populations have TB rates as much astwo to three times higher than the nationalTB rate (data from the Venezuelan NationalTB program).

To combat TB there is a need for newtherapeutics, diagnostics and vaccines, inconjunction with improved operationalguidelines to enhance current TB controlstrategies (3). The only markers availableto study TB epidemiology in the 1980’swere drug susceptibility profiles and phagetypes (4-6), but since the 1990’s new mark-ers and new methods have opened the

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TB in Sucre 333

doors to the molecular epidemiology of M.

tuberculosis and made it possible to under-stand the population structure of this suc-cessful pathogen (7-15). Particular strainsor strain families have been found to be dis-proportionate contributors to both localand global TB, and the careful study ofdominant compared to rare strains shouldprovide clues to the determinants of theirsuccess and possibly yield strategies forcontrol (16, 17).

Molecular epidemiological analysis ofTB epidemics in different populations usu-ally encounters a number of phylogeneti-cally unrelated lineages (18). For patho-genic microorganisms, fitness generally re-fers to heritable traits among members of agiven species or phylogenetic lineage thatdetermine its relative growth or pathogeniccapacity compared to all other members(3). Different clinical M. tuberculosis

strains can induce different host immuneresponses, as well as differences in relativepathogenesis and virulence that can bedemonstrated in animal models (19-22).Knowledge of the local M. tuberculosis epi-demiology and molecular epidemiologyshould help to define outbreaks and identifydominant clones that could be more viru-lent, more easily transmissible or drug re-sistant, and which may be amenable to tai-lored strategic interventions to controltheir spread.

We previously characterized the molec-ular epidemiology of M. tuberculosis strainsisolated in diverse regions of Venezuela, in-cluding many strains isolated from the mar-ginal neighborhoods in the western part ofthe capital Caracas (23). We found domi-nant Shared International Types (SITs) byspoligotyping and identified a few highlyprevalent genotypes by MIRU-VNTR analysis.Caracas, the capital of Venezuela, is a city ofabout 6 million inhabitants with several geo-graphically distinct marginal regions. In thepresent work we sought to extend our study

to the eastern part of Caracas located inthe Sucre municipality of the Mirandastate, a very large, densely populated mar-ginal area with about a 1,133,000 inhabit-ants, many with low socioeconomic status,and more tuberculosis cases per year thanany other municipality in the country. Theaim of this work was to determine whichneighborhoods have the greatest burden oftuberculosis cases and to delineate the pop-ulation structure of M. tuberculosis strainscirculating in the Sucre municipality. Fur-thermore, we sought to determine whetherthe distribution of strain families is differ-ent from that previously found in the west-ern part of Caracas, and whether particulargenotypes predominated in different neigh-borhoods, commonly termed “Barrios,” ofthe Sucre municipality.

MATERIALS AND METHODS

Cases 2005-2006

To determine the sectors of Sucre mu-nicipality with the most cases of tuberculo-sis, a retrospective study was undertaken toidentify all of the residents of this munici-pality who had been diagnosed with tuber-culosis in 2005-2006. This was done bysearching the records of: the tuberculosiscontrol program of Health District #7,which includes Sucre municpality; the Ven-ezuelan National TB Control Program; theTB diagnostic laboratory of the VenezuelanNational Institute of Hygiene; and the Tu-berculosis Laboratory of the Institute ofBiomedicine in Caracas. A total of 981cases were identified, 492 diagnosed in2005, and 489 in 2006. Of these, it was pos-sible to determine residence within a par-ticular sector or Barrio of Sucre municipal-ity for 829 cases.

Clinical isolates

A preliminary molecular epidemiologystudy performed in 2006 included a conve-

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334 Patiño y col.

nience sample of 44 available M. tuberculo-

sis strains from patients with pulmonary tu-berculosis isolated from January-July 2006,either in the bacteriology lab of the PetareMaternal-Infant Hospital “Joel ValenciaParpacén,” or in the National MycobacterialReference lab. The strains were spoligo-typed and categorized by the Barrios listedas the patients’ residence (Table I).

In 2009-2010, a more extensive molec-ular epidemiology study was performed. Thestrains analyzed were a convenience sampleof 131 available M. tuberculosis strains iso-lated from 2006 to 2010. All patients werefrom municipality Sucre and diagnosed ei-ther in the Laboratory Luis Quiñones, lo-cated in the annex of the Petare Mater-nal-Infant Hospital, or the TB Laboratory ofthe Instituto de Biomedicina. Sputum sam-ples were treated with the Petroff method(24) and inoculated onto LowensteinJensen slants, or with the Kudoh Methodand inoculated onto Ogawa slants (25), andincubated at 37°C for 3 to 6 weeks untilcolonies were visible. Patient location andstrain distribution were as follows: Petare29, Unión 19, San Blas 13, Maca 11,Carpintero 7, José Félix Rivas 6, Filas de

Mariche 5, Mirador del Este 5, Agricultura3, 5 de Julio 2, Antonio José de Sucre 2, ElCampito 2, Isaías Medina 2, La Línea 2, SanIsidro 2, Guaicoco 2, La Bombilla 2, La Cal-ifornia 2, La Dolorita 2, 19 de Abril 1,Bolívar 1, Cuatricentenario 1, El Dorado 1,El Nazareno 1, San Miguel 1, Vista Alegre 1,Boleíta 1, Buena Vista 1, Campo Rico 1, ElLlanito 1, La Urbina 1 and Turumo 1(Fig. 1 and Table I).

DNA extraction

Briefly, 2 to 3 loopfulls of coloniesfrom original slants were resuspended in250 µL of Tris- EDTA 1X buffer and heatedat 95°C for 15 minutes in 1.5 mL plastictubes. The tubes were then centrifuged at13.000 rpm and 5-10 µL of the supernatantwas used for each PCR reaction.

Spoligotyping

The CRISPR region (26, 27) was usedas a target for membrane-based Spoligo-typing, as previously described (9, 23, 28).The hybridization patterns for the 43spoligotype spacers were transcribed fromthe films into a previously formatted and la-beled Excel spreadsheet and double-

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TB in Sucre 335

Fig. 1. A. Map; and B. Satellite photo of municipio Sucre in Miranda state, showing the sectors or“Barrios” with the highest number of cases of tuberculosis. The numbers below names of thesectors indicate the percentage of strains from that sector included in the molecular epidemi-ology study of 131 isolates/the percentage of TB cases diagnosed in patients from that sectorin 2005-2006, out of the total 839.

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TB in Sucre 337

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checked to insure the accuracy of the resul-tant table (23). Spoligotypes were com-pared with the most recent release of theInternational Spoligo Database, SITVITWEB(29). Our national Mycobacterium tubercu-

losis database (MTBdbase) was used forstrain comparisons in order to find geno-types circulating in Venezuela, andSPOTCLUST (http://tbinsight.cs.rpi.edu/run_spotclust.html) (30), was used to ten-tatively assign phylogeographic lineages tospoligotype patterns not reported inSITVITWEB.

Homemade membranes for hybridization

Byodine-C Nylon membranes were pre-pared for spoligotyping as previously de-scribed (9). The oligonucleotide probeswere generally the same as described (9),with some corrected sequences (31). Home-made membranes were subject to qualitycontrol measures to insure that adequatehybridization signals could be obtainedfrom all spacer oligonucleotides fixed to themembranes.

MIRU-VNTR genotyping

Mycobacterial interspersed repeti-tive-unit-variable-number tandem-repeatanalysis was performed on strains belongingto the principal clusters defined by spoligo-typing to accurately establish strain iden-tity. Fifteen loci were amplified using multi-plex PCR and run with Rox labeled MapMarker 1000 size standards (Bioventures)on an ABI3130xl sequencer as describedpreviously (26). Sizing of the PCR frag-ments and assignment of MIRU- VNTR al-leles were done with the Gene Mapper soft-ware package (PE Applied Biosystems).

Dendrogram

The online MIRU-VNTRplus tool (32,33) was used with the UPGMA algorithm(34) for cluster analysis. The UPGMADendrogram treated the VNTR values as

categorical data (35) and the spoligotypingdata was treated as binary with theJaccard’s index (36).

RESULTS

This report describes three separatestudies of the traditional and molecular epi-demiology of tuberculosis in the Sucre mu-nicipality of the Miranda state of Venezuela.The first study retrospectively searched therecords of the local and national TB controlprograms, as well as the diagnosticmycobacteriology labs in Caracas and found829 patients who were diagnosed with tu-berculosis in 2005-2006 and had an addresswithin the municipality. A pilot molecularepidemiology study performed spoligo-typing on 44 strains isolated in 2006 frompatients living in the municipality, and thena larger study on molecular epidemiologyanalyzed 131 M. tuberculosis clinical strainsisolated from Sucre residents between2006-2010. The distribution of the cases bythe sector, or “Barrio” of residence in themunicipality is shown in Table I and Fig. 1.Combining the three studies, the patientscame from 80 different geographic sectorsof the municipality, indicating that tuber-culosis appears to be an endemic problemin the entire municipality. In all three stud-ies, however, more than 45% of the casesresided in the same sectors: Petare, Unión,San Blas, Maca, Carpintero, José FélixRivas, Filas de Mariche and Mirador delEste. Fig. 1A is a conventional map of theregion, and Fig. 1B is a satellite map of themunicipality (Google maps), showing thepercentages of cases from the two largerstudies that resided in the different sectors.From the satellite image it is clear that themost affected Barrios are also the largestand most densely populated. Petare was thesector most commonly listed as place ofresidence in the two molecular epidemiol-ogy studies, with 20.5% and 22.1% of cases

338 Patiño y col.

respectively (Table I), but this may not beaccurate, as Petare is not a large Barrio butis a name commonly used to non-specifi-cally indicate the marginal, low resourcesectors of Sucre municipality, and may havebeen written in the patient records whenthe precise sector of residence in the mu-nicipality was unknown.

In the pilot study of 44 strains, therewere 22 spoligotypes, of which 3 were notpresent in SITVITWEB. There were 4 clus-ters: SIT17, 4 strains; SIT42, 7 strains;SIT20, 3 strains; and SIT1692, 2 strains(Table IV).

In the study of 131 strains, there were41 different spoligotypes (Table II), ofwhich 27 were in the internationalspoligotype database SITVITWEB. The re-maining 14 spoligotype patterns, represent-ing 18 patients (14%), were not in the data-base, but SPOTCLUST determined thatmost belonged to the LAM (Latin AmericanMediterranean) lineage. The isolatesgrouped into 15 spoligotype clusters with 2to 27 strains each, representing a total of105 (80%) strains (Table II). Thirteen ofthe clusters had spoligotypes present inSITVITWEB, while the spoligotypes of theremaining two groups, composed of 4 and 2strains respectively, were not inSITVITWEB. SPOTCLUST assigned them tothe T1 and LAM9 lineages respectively. Ofthe 14 spoligotypes not assigned SITs, fivewere previously submitted to SITVITWEB,one from Senegal and four that were iso-lated in a prior survey of Venezuelan strainsby Aristimuño et al (37).

Table III shows the percentages of thedifferent SITpatterns in the two molecularepidemiology studies of municipality Sucrestrains, compared to the previously pub-lished percentages of the SITpatterns instrains isolated in the Instituto de Biome-dicina located in western Caracas and thestates of Carabobo, Delta Amacuro,Amazonas and Sucre. In both molecular ep-

idemiology studies, SIT’s 42 and 20 aremore common in municipality Sucre thanin any other region, including the westernside of Caracas.

Table IVA shows the distribution bysector of the 26 clustered isolates in thesmaller study, and Table IVB shows the dis-tribution by sector of the 105 clusteredspoligotypes from the study of 131 isolates.The four most commonly isolatedspoligotypes, SITs 17, 42, 93 and 20, all be-longing to the Latin American Mediterra-nean (LAM) lineage, were found in patientsfrom at least 7 different Barrios. The mostfrequent spoligotype pattern in the munici-pality, SIT17, was found in 12 different Bar-

rios. In the larger study the Unión sectorhas 7 of the 19 SIT42 clustered strains,while Petare had 4 and Maca 3 isolates. Inthe smaller study Unión had 2 of the 7SIT42 strains, the only sector with morethan one SIT42 isolate. Of the SIT20 clus-ter in the larger study, 4 of 9 were fromUnión and 2 from San Blas. There was alsoa cluster of 3 SIT20 strains in the smallerstudy, one from each of Petare, Palo Verdeand the Colonial region of Petare.

Although several of the clusteredspoligotypes were isolated from more thanone patient in particular Barrios, spoligo-typing is not sufficiently discriminative todetermine whether this was evidence of lo-cal spread of a specific strain within theBarrio. Therefore, in the larger molecularepidemiology study we used 15 MIRU-VNTRloci to analyze a sample of 21 spoligotypedstrains from the largest spoligotype clus-ters, SIT17 and 42, and also 4 clusteredstrains with spoligotype patterns not pres-ent in the SITVITWEB database. Adendrogram built with the results (Fig. 2)showed that 4 of the SIT42 isolates fromBarrio Unión had an identical MIRU-VNTRpattern that was not seen in previous stud-ies of other regions of Venezuela. ThisSIT42 MIRU-VNTR pattern was also not

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TB in Sucre 339

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present in the MIRU-VNTRplus database,where the most similar SIT42 strain dif-fered at 5 of 15 MIRU-VNTR loci. Threeother SIT42 strains from the same Barrio

were closely related, differing by only one ortwo loci (strains 10, 11 and 17). An addi-tional three very closely related strainsfrom Maca (14, 15 and 19), differ fromthose in Unión only at locus 4052.

Four of the SIT17 strains—two fromBarrio Jose Felix Rivas (6 and 7), one fromFilas de Mariche (5) and one from Barrio

Carpintero (3)—had MIRU-VNTR patternsthat were identical to a large cluster previ-ously found to be widely distributed in Ven-ezuela. Another SIT17 strain from Filas deMariche (5) differed only at locus 960, andone from Barrio Carpintero (1) was differ-ent only at two loci - 580 and 4156.

Two strains from Maca with an identi-cal spoligo pattern, not found inSITVITWEB (Fig. 2 strains 18 and 21), hadrelated MIRU-VNTR patterns that varied injust two loci-802 and 1955.

DISCUSSION

This study found that the distributionof SITs in the strains of M. tuberculosis cir-culating in the large marginal region in theeast of Caracas-Sucre municipality was sim-ilar to the distribution previously reportedfor the west of Caracas and other parts ofVenezuela, showing a wide diversity of M.

tuberculosis genotypes, but differed in hav-ing greater proportions of SIT42 and SIT20strains. The MIRU-VNTR analysis found thatthe SIT42 strains appeared to be related,

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TB in Sucre 345

Fig. 2. UPGMA dendrogram built with the MLVA and Spoligotype information using the MIRU- VNTRplus site. Each code is made of a composition of the arbitrary position number that was takenfrom a file and then used for the tree, the strain code itself, the SIT code, the Lineage identifi-cation and the patient location.

and were found principally in the Unión andMaca Barrios. In addition, there werestrains that belong a previously identifiednationwide SIT17 cluster, which were moreprevalent in the Barrios Carpintero, JoseFelix Rivas and Filas de Mariche.

In our previous study of 1298 strainsfrom the western region of Caracas and 6geographically distributed states (23), themost common spoligotype patterns (TableIII) were: SIT17 (18.6%); SIT93 (9.9%);SIT605 (7.2%); SIT42 (6.5%); SIT53 (4.1%);SIT20 (2.7%); and SIT50 (1.7%) (23). Theoverall distribution was skewed, however, bya very large number of SIT605 strains inCarabobo, where it was the most commonSIT found, while it was much less commonin all other regions, including Sucre munic-ipality. The two molecular epidemiologystudies of strains from Sucre municipalityshowed some differences in the prevalenceof different SITs but basic strain distribu-tion was generally similar to the other re-gions of Venezuela studied, with SIT17 themost common spoligotype (Table III). Sur-prisingly though, both studies showed thatSIT42 and SIT20 were more common inSucre municipality than in any other regionof Venezuela studied, and the percentagesin the two studies were remarkably similar:14.5% and 15.9% for SIT42; 6.9% and 6.8%for SIT20. This was the only region we haveexamined where SIT42 is the second mostcommon pattern (Table III), and the per-centages of strains with SIT42 and SIT20 inthis municipality were twice that found instrains isolated in western Caracas.

When the distribution of strainspoligotypes was examined by Barrio, it ap-peared that some clustered strains weremore common in particular Barrios, butspoligotyping is not sufficientlydiscriminative to identify transmission oroutbreaks because the identical spoligo-types can be shared by strains with differentgenotypes. Therefore, a limited number of

the clustered strains were examined by themore discriminative technique of MIRU-VNTR 15 loci. We previously (23) found alarge MIRU-VNTR cluster of a SIT17 strain(261423442122333) that was distributedthroughout the country, and 5 strains fromSucre municipality belonged to this cluster,two from Filas de Mariche, two from JoseFelix Rivas, and one from Carpintero, and asecond strain from Carpintero was differentin only two MIRU-VNTR loci.

The most striking finding was that theeleven SIT42 strains studied by MIRU-VNTR15 were all closely related. Four strains iso-lated from patients living in Barrio Uniónhad an identical MIRU-VNTR pattern,251423242122434, which had not beenpreviously found in Venezuela. One addi-tional strain differed from these four identi-cal strains by one locus, another by twoloci, and a third by 3 loci. Three otherstrains differing by only two loci came fromBarrio Maca, and one of the differences, atMIRU-VNTR locus 2163b, was the same inall of these three strains, suggesting thatthey may be an offshoot strain circulatingin Maca. A strain with three differences wasfrom Jose Felix Rivas. Except for Petare,the only Barrios with more than one SIT42isolate were Unión and the adjacent Maca(Fig. 1), so the spread of this and closely re-lated strains, especially in these Barrios,could account for the higher prevalence ofSIT42 in the Sucre municipality comparedto the rest of the country. There were 4SIT42 strains isolated in Petare, but, asmentioned above, “Petare” is a generalterm for the marginal neighborhoods inSucre municipality, and is often listed asresidence when the correct sector or Barrio

is unknown; the actual geographic region“Petare” does not contain a large marginalpopulation. It is thus possible that some ofthese 4 “Petare” SIT42 strains might alsobe from patients living in Unión or Maca.They were not analyzed by MIRU-VNTR 15,

346 Patiño y col.

so it is not known if they share the geno-type of the SIT42 strains from Unión.

From these small samples of strains, itappears that perhaps 20-25 % of the tuber-culosis in Sucre municipality is caused bydominant strains of SIT17 and SIT42 andtheir offshoots, and that the SIT42 clusteris a strain particular to this municipalitythat is being actively transmitted, especiallyin the densely populated region of Uniónand the adjacent Barrio Maca. The presenceof strains with differences at 2-3MIRU-VNTR loci suggests that the SIT42strain may have been present and circulat-ing in the municipality for many years.Unión and Maca are home to many immi-grants from Colombia and other SouthAmerican countries, who often live clus-tered in country-specific sectors of theseBarrios. The SIT42 strain circulating inthese Barrios was not found in other partsof Venezuela and could have been intro-duced by an immigrant and spread withinthe immigrant’s community. ThisMIRU-VNTR pattern is not similar to anystrain in the MIRU-VNTRplus database, sowithout more detailed epidemiologic inves-tigation, it is not possible to speculate on apossible country of origin. A recent reportdescribed the predominance of SIT42 in theneighboring country of Colombia (38), butnone of the Colombian isolates had VNTRpatterns similar to that of the SIT42 clusterin Sucre municipality (Dr. Jaime Robledo,personal communication).

The presence of strains belonging tothe large nationwide SIT17 cluster is per-haps further evidence that this strain mayhave some advantage in virulence ortransmissibility over others circulating inVenezuela (23). The high percentage ofSIT20 strains in this municipality is also in-teresting, but we were not able to performMIRU-VNTR analysis on them to see if theybelong to a single dominant genotype. Itappears that the extensive marginal areas

of Sucre municipality, such as Unión, areregions with that have had a persistent,long-term tuberculosis presence resultingin the microevolution of their own family ofstrains, especially SIT42, that are not foundin other regions, not even in the large mar-ginal regions of western Caracas. As can beseen in Fig. 1, Barrio Unión is at the centerof a very large, densely populated region,and may be relatively isolated.

This study found a large diversity ofspoligotype patterns, many of which werenot found in the SITWEB international database or previously seen in Venezuela, andwere identified in only one isolate from theSucre municipality. Although it is possiblethat technical errors were responsible forthis diversity, this seems unlikely, as all iso-lates were tested at least twice on differentmembranes and the results were consistenton more than one reading. Strains that dif-fer by just one or two MIRU-VNTR differ-ences were regarded as the same genotype,but recent studies using whole genome se-quencing found that these strains can beonly distantly related (39). However, as thestrains were from patients living in the samegeographic region and had patterns that dif-fer only slightly from a predominant pattern,a process of microevolution seems likely.

From our retrospective search forcases diagnosed in 2005-6, as well as datafrom more recent records of the TB controlprogram, it appears that each year thereare between 500 and 600 new cases of tu-berculosis in Sucre municipality, yieldingan incidence of about 45-50 cases per100,000 inhabitants per year. This figure islikely to be an underestimate because notall cases are diagnosed, although it is diffi-cult to calculate an accurate incidence be-cause of the fluid and growing nature of thepopulation. Nevertheless, the true inci-dence is probably significantly higher thanthe overall estimated TB incidence in Vene-zuela of ~33/100,000.

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TB in Sucre 347

This study had several shortcomingsand limitations. The sample of 131 strainspresented here represents only about aquarter of one year’s cases, and the earliercohort of 44 strains represents less than atenth. These were convenience samples ofthe available strains isolated from patientspresenting to the municipality’s only TBclinic, and we have no way of assuring theyare accurately or proportionately represen-tative of TB cases in the municipality. Infact, there is evidence that our molecularepidemiology study of 131 cases was notproportionately representative, becauseJose Felix Rivas is a very large, densely pop-ulated Barrio that was represented withonly 5 cases in this cohort. While the Bar-

rios with the most TB cases are generallythe most densely populated in the munici-pality, we don’t have accurate populationdata for these Barrios to determinewhether the TB incidence is higher in thesethan in other sectors of the municipality.However, in the larger study of all casesfrom 2005-6, Jose Felix Rivas had manymore cases than any other sector, presum-ably reflecting its very large population andcrowded housing. It is unlikely that changesin the incidence of tuberculosis in this bar-

rio over a few years could account for thisdifference, so the molecular epidemiologystudy with 131 isolates may haveoversampled Barrio Unión, which had thehighest percentage of TB cases (14.5%) inthat study. It is also possible that this re-flects an ongoing outbreak of the SIT42strain in this sector, or a tendency for pa-tients from Jose Felix Rivas to be diagnosedelsewhere.

The molecular epidemiology studieswould have been more complete if the co-horts were larger and all of the strains clus-tered by spoligotyping could have been ana-lyzed by MIRU, but this was not possibledue to limitations of time, resources andpersonnel. It would also be worthwhile look-

ing at the residency data for those patientswith similar or identical MIRU-VNTR pat-terns, to see if they live in close proximitywithin the Barrios, or belong to the samefamilies or social groups. Furtherepidemiologic investigation in the high in-cidence Barrios might reveal that patientsinfected with the same strains have linksamenable to strategic interventions to in-terrupt the on-going transmission, but thiswas not possible within the scope and re-sources for this study. These sectors are dif-ficult to reach and challenging for outsid-ers. A more feasible approach might to re-ducing the incidence might be to work withcommunity groups within the highest prev-alence sectors to promote the early diagno-sis of cases, the search for epidemiologicassociations, investigation of their closecontacts, and outreach to patients whoabandon therapy before its completion.

ACKNOWLEDGMENTS

This work would not have been possi-ble without the efforts of CorpoMir to buildthe TB diagnostic laboratory “LuisQuiñones-TB Sucre”. The authors acknowl-edge the important contributions of thestaff of the laboratory “Luis Quiñones–TBSucre” in diagnosing tuberculosis in themunicipality and providing cultures of thepatient isolates; the direction of Health Dis-trict #7; and the director and maintenancestaff of the Hospital Materno-Infantil dePetare “Joel Valencia Parpacén”. The au-thors also thank Sandra Fernández and theVenezuelan National Institute of Hygiene,and Carmen Ramirez and the National Tu-berculosis Reference lab for contributingstrains analyzed in this study. This work wassupported by FONACIT projects2001001851 and G02005000393, as well asLOCTI projects “Tuberculosis en Vene-zuela,” and “Programa IntegradoComunitario para el Control de la Tubercu-

348 Patiño y col.

losis en el Municipio Sucre del EstadoMiranda.

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