INT J TUBERC LUNG DIS 17(2):246–250 © 2013 The Union http://dx.doi.org/10.5588/ijtld.12.0195
Spoligotyping of multidrug-resistant Mycobacterium tuberculosis isolates in Ethiopia B. Diriba,*† T. Berkessa,†‡ G. Mamo,§ Y. Tedla,¶ G. Ameni† * St Paul’s Hospital Millennium Medical College, Federal Ministry of Health, Addis Ababa, † Aklilu Lemma Institute of Pathobiology, Addis Ababa University, Addis Ababa, ‡ Sheki Health Centre, Jimma, § School of Veterinary Medicine, Addis Ababa University, Debre Zeit, ¶ St Peter TB Specialized Hospital, Addis Ababa, Ethiopia SUMMARY SETTING:
St Peter Tuberculosis (TB) Specialized Hospital and the Aklilu Lemma Institute of Pathobiology, Addis Ababa, Ethiopia. O B J E C T I V E : To genotype multidrug-resistant tuberculosis (MDR-TB) isolates and assess the magnitude of their clustering. D E S I G N : A total of 183 consecutive MDR-TB isolates collected between September 2009 and February 2012 were characterised using molecular typing. Prior to the study, the isolates were confirmed as MDR-TB using GenoType® MTBDRplus. Recent transmission index was used to analyse the clusters. R E S U LT S : Spoligotyping identified 43 different patterns, of which 17 consisted of at least two isolates forming clusters, while 26 had only a single isolate. The
most frequent patterns were spoligo international typing (SIT) number 21 and 149. Twenty-four patterns did not match existing patterns in the SpolDB4 database. The strains belonged to three lineages, the predominant lineages being Euro-American and Indo-Oceanic, each consisting of 65 isolates. High proportions (86%) of patients were infected with clustered strains, suggesting probable recent transmission of MDR-TB in the study area. C O N C L U S I O N : The observation of cluster formation of the spoligotype patterns of MDR-TB isolates could suggest transmission of MDR-TB strains among the population, thus warranting further attention. K E Y W O R D S : drug resistance; recent transmission; clustering; lineage; tuberculosis
TUBERCULOSIS (TB) is the leading cause of morbidity and mortality worldwide due to infectious disease, with a major impact in developing countries. Ethiopia ranks eighth among the 22 high TB burden countries in the world, and the third in Africa.1 According to the 2010 World Health Organization (WHO) report, the prevalence, incidence and mortality of TB (all forms) in Ethiopia is estimated to be respectively 572, 359 and 64 per 100 000 population.2 The emergence of multidrug-resistant (MDR-) and extensively drug-resistant (XDR-) TB is one of the reasons for the increasing problem, posing a serious threat to the worldwide control of the disease.3–5 MDR-TB is defined as TB disease caused by Mycobacterium tuberculosis strains resistant to the two most powerful anti-tuberculosis drugs, isoniazid (H, INH) and rifampicin (R, RMP), while XDR-TB is defined as MDR-TB plus resistance to a fluoroquinolone and at least one second-line injectable agent (amikacin, kanamycin and/or capreomycin).6 Ethiopia is also one of the 27 countries with the highest number of MDR-TB cases in the world. In
2009, MDR-TB cases constituted 1.6% of new and 12% of previously treated TB cases;7 and according to the 2011 WHO report, there were approximately 2100 cases of MDR-TB in Ethiopia.8 The design of strategies for the management of MDR-TB depends on an understanding of the development and spread of resistant isolates. Mycobacterial strain typing using molecular methods has become an important tool for TB surveillance, control and prevention.9 Spoligotyping is a genotyping method used to study the epidemiology of the M. tuberculosis complex.10 As with other molecular markers, spoligotyping enables classification of isolates into distinct clusters, and thus allows characterisation of the genetic diversity of M. tuberculosis. Spoligotyping has been found to be easy, rapid and suitable for use in computer-assisted analysis of many molecular patterns at the same time, thus allowing its use in large-scale epidemiological surveys.11,12 However, spoligotyping has shown a discriminatory ability that is lower than that of insertion sequence (IS) 6110 restriction fragment length polymorphism analysis, with the
Correspondence to: Gobena Ameni, Animal Health and Zoonoses, Aklilu Lemma Institute of Pathobiology, Addis Ababa University, PO Box 1176, Addis Ababa 1176, Ethiopia. Tel: (+251) 911 763 091. Fax: (+251) 127 552 96. e-mail:
[email protected] Article submitted 12 March 2012. Final version accepted 10 September 2012.
MDR-TB spoligotyping in Ethiopia
exception that it has the specific advantage of higher discrimination of strains with low copy numbers (⩽6) of IS6110.13–15 Spoligotyping is currently proposed as the initial screening step in a multistep typing strategy for epidemiological studies.12,15 Although Ethiopia is one Africa’s high TB prevalence countries, there is limited information on the genotypic characteristics of M. tuberculosis. The availability of such information would help to study the phylogenetic characteristics of the organism, which in turn could provide new insight into the natural history of TB.16 Spoligotyping of MDR-TB is thus important in providing a better understanding of the various aspects of MDR-TB epidemiology and transmission. The objective of this study was to genotype MDRTB isolates and assess their magnitude in selected regions of Ethiopia.
MATERIALS AND METHODS Source of the isolates One hundred and eighty-three consecutive MDR-TB isolates collected between September 2009 and February 2012 at St Peter TB Specialized Hospital and confirmed to be RMP- and INH-resistant using the GenoType® MTBDRplus assay (Hain Lifescience, Nehren, Germany) were included in the study. St Peter Hospital is a national TB referral hospital, and its TB laboratory serves as a mycobacteriological reference centre for MDR-TB in Ethiopia. Before characterisation using the molecular method, the stored colonies were subcultured at St Peter Hospital, they were then harvested and killed by heating at 80°C for 1 h, after which they were transported to the Aklilu Lemma Institute of Pathobiology (ALIPB) for molecular typing. Subculturing of stored isolates The isolates were first frozen in BACTECTM MGITTM 960 Tubes (BD, Sparks, MD, USA) at –80°C, and then removed and thawed at room temperature. Thereafter, 0.1 ml of the suspension was spread on LöwensteinJensen (LJ) slants. The slants were incubated aerobically at 37°C for 6–8 weeks, with weekly observation of the growth of colonies. Microscopic examination of culture using Ziehl-Neelsen staining was performed to confirm acid-fast bacilli-positive isolates. Heatkilled cells of each isolate were prepared by mixing loopfuls of cells in 200 μl distilled water; the cells were killed by heating at 80°C for 1 h, after which they were used for molecular typing. RD9 deletion typing Region-of-difference 9 (RD9) deletion typing was performed on heat-killed cells to confirm the presence or absence of RD9, as described earlier,17 using RD9 flankF, IntR and flankR primers, each at a concentration of 100 μM. Polymerase chain reaction (PCR) amplification was performed on each sample
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using a standard thermo cycler (VWR Thermo cycler, VWR International, East Grinstead, UK). The PCR amplification mixtures used for RD9 typing were as follows: the reaction mixture consisted of 10 μl HotStarTaqMaster Mix (Qiagen, Crawley, UK), 7.1 μl distilled water, 0.3 μl of each of the three primers (100 mM) and 2 μl DNA template (heat-killed cells), giving a total volume of 20 μl. Gel electrophoresis was used for the separation of the PCR product. For gel electrophoresis, 8 μl PCR product was mixed with 2 μl loading dye, loaded onto 1.5% agarose gel and electophoresed at 100 V and 500 mA for 45 min. The gel was then visualised using a computerised MultiImage Light Cabinet (VWR). M. tuberculosis H37Rv, M. bovis bacille Calmette-Guérin, and water were included as positive and negative controls. Interpretation of the result was based on bands of different sizes, as previously described by Parsons et al.17 Spoligotyping Spoligotyping was performed as previously described by Kamerbeek et al.10 and as per the spoligotype kit supplier’s instructions (Ocimum Biosolutions, Ijsselstein, The Netherlands). The direct repeat (DR) region was amplified by PCR using oligonucleotide primers derived from the DR sequence. A total volume of 25 μl of the following reaction mixture was used for the PCR: 12.5 μl of HotStarTaq Master Mix (Qiagen; this solution provides a final concentration of 1.5 mM MgCl2 and 200 mM of each deoxoribonucleotide triphosphate), 2 μl of each primer (20 pmol each), 5 μl suspension of heat-killed cells (approximately 10–50 ng), and 3.5 μl distilled water. The amplified product was hybridised to a set of 43 immobilised oligonucleotides, each corresponding to one of the unique spacer DNA sequences within the DR locus. Hybridised DNA was detected by the enhanced chemiluminescence method (Amersham Biosciences, Amersham, UK) and by exposure to X-ray film (Hyperfilm ECL, Amersham Biosciences), as specified by the manufacturer. Recent transmission index (RTI) was estimated using the formula proposed by Small et al.,18 i.e., RTIn − 1 and RTIn, which considers the number of patterns with a unique genotype (singletons), as described by Luciani et al.19 Ethical clearance The research proposal was reviewed and given ethical approval by the Institutional Review Board of the ALIP, Addis Ababa University and the St Peter TB Specialised Hospital Ethics Review Committee.
RESULTS Demographic characteristics of patients The demographic data of the patients from whom the MDR-TB isolates were obtained are shown in the Table. The patients were from 10 regions of the
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Table Demographic data for the tuberculosis patients from whom the MDR-TB M. tuberculosis isolates were obtained Variable Sex Male Female Age, years 15–24 25–34 35–44 ⩾45 History of anti-tuberculosis treatment Previously treated Not previously treated Region Addis Ababa Oromia Southern Regions (SNNPR) Tigray Somali Amahara Diredawa Gambella Affar Benshangul
Frequency n (%) 100 (54.6) 83 (45.4) 67 (36.6) 67 (36.6) 35 (19.1) 14 (7.7) 180 (98.4) 3 (1.6) 123 (67.2) 24 (13.1) 12 (6.6) 7 (3.8) 9 (4.9) 3 (1.6) 2 (1.1) 1 (0.6) 1 (0.6) 1 (0.6)
MDR-TB = multidrug-resistant tuberculosis; SNNPR = Southern Nations, Nationalities, and People’s Region.
country; 98.4% had been previously treated with first-line anti-tuberculosis drugs. RD9 deletion typing The presence of RD9 was confirmed in 182 of the 183 isolates, indicating that they were M. tuberculosis; the remaining isolate did not give a signal in response to RD9 typing. Spoligotyping A total of 43 distinct spoligotype patterns were identified from the 183 isolates analysed using spoligotyping (Figure): 157 (85.79%) were grouped in 17 clusters of spoligotype patterns. Of the 123 isolates from Addis Ababa, 102 (83%) formed clusters, while 55 (91.7%) of the 60 isolates from other regions formed clusters. Overall, two thirds of the clustered strains were identified from Addis Ababa. Patients with unrelated spoligotypes were assumed to have different strains of M. tuberculosis and therefore to have acquired the disease from unrelated sources. Clustered patients were likely to have related strains of M. tuberculosis, which might have arisen from a common or related source. Taking the number of cases with unique genotypes (singletons) into account, RTIn was 0.86, while RTIn − 1 was 0.76 when a single isolate was considered as the cause of a cluster. The largest cluster identified in the present study, spoligo international typing (SIT) no. 21, consisted of 32 isolates; the second largest cluster was SIT149, comprising 27 isolates. SIT523 and SIT54 comprised respectively 23 and 17 isolates. Nevertheless, 26
(14.2%) clinical isolates were represented by a unique (non-clustered) spoligotype pattern. In addition to these patterns, which are already recorded in the fourth international spoligotyping database (SpolDB4), there were 24 previously unreported spoligopatterns, new to SpolDB4. Among these new spoligotypes, 18 were represented by unique patterns, each consisting of a single isolate, while the remaining six consisted of clusters of isolates: the largest pattern consisted of six isolates, while the remaining five patterns consisted of two to five isolates. The spoligotype families identified in this study were T, CAS, U, MANU, Haarlem (H) and East African Indian (EAI), at respectively 27.3%, 24.6%, 12.6%, 10.9%, 1.6% and 1.09%. However, the 24 new patterns, comprising 40 (21.9%) isolates, could not be classified. Further identification of the new patterns using the SpotClust programme indicated that the most likely families were Family 33, T, CAS, EAI and Family 34, consisting of respectively 17, 12, 8, 2 and 1 isolates. Classification of the strains by lineage showed three different M. tuberculosis lineages; the most prevalent were Euro-American and Indo-Oceanic (65 isolates each), followed by EAI, consisting of 53 isolates.
DISCUSSION In recent years, molecular fingerprinting techniques have been increasingly employed to define the relationship between mycobacterial strains and clarify the epidemiology of TB transmission.20 In the present study, 183 MDR-TB strains isolated at St. Peter TB Specialised Hospital were characterised using spoligotyping to determine the level of clustering among MDR-TB patients in selected regions of Ethiopia. The families and lineages of the strains were identified. On spoligotyping the 183 MDR-TB isolates, a total of 43 different spoligotype patterns were identified. A previous study in Ethiopia showed that SIT149 and SIT21 were the first and second most frequently clustered strains among MDR-TB isolates;21 however, SIT21 was the predominant strain found in the present study. SIT21 is characterised by the absence of spacers 4–7, 10 and 20–35, and is the most prevalent strain in East Africa.22 It has been reported in 14 countries: Australia, Belgium, Denmark, Ethiopia, Finland, France, Gabon, Germany, Italy, Libya, Netherlands, Norway, Sweden and the United States. The second most frequent pattern, SIT149, characterised by the absence of spacer 10–19 and 33–36, has been reported in 22 countries. Although SIT523 was the third most frequent strain in our study, it was not identified in a previous study. SIT523 is characterised by the presence of all 43 spacers, and it has so far been reported in 11 countries. The number of SIT523 isolates in this study was four isolates higher than the number registered in SpolDB4. This might be due to
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Figure Spoligotype patterns of M. tuberculosis isolates from pulmonary tuberculosis patients. Filled boxes = presence of spacers, empty boxes = absence of spacers. The largest cluster was SIT21, consisting of 32 isolates, and SIT149, consisting of 27 isolates. SIT523 and SIT54 had respectively 23 and 17 isolates. Nevertheless, 26 (14.21%) clinical isolates were represented by a unique (nonclustered) spoligotype pattern. Twenty-four new spoligotype patterns were identified for the first time, and were new to the international database, spolDB4. Classification of the strains on the basis of lineage showed three different lineages of M. tuberculosis, the predominant lineages being Euro-American and Indo-Oceanic. SIT = spoligo international typing; SpolDB4 = fourth international spoligotyping database.
the association of this ancestral strain with East African countries. This strain may have affected early hominids in the East African region around 3 million years ago.23 It could also be due to mixed infection, which needs further investigation using more powerful molecular techniques. This was not possible in the present study setting, due to the lack of molecular typing facilities with greater discriminatory power than that of spoligotyping. Cluster formation was higher in regions outside Addis Ababa. This may be due to the fact that Addis Ababa, as the capital of the country, has a mixed population from different regions of the country. As
indicated earlier, the mixing of populations from different regions leads to fewer cluster formations.24 Considering the probable existence of an index case in each cluster, it was estimated that 76% (157 − 17 = 140) of the 183 cases may have been due to recent infection.18 Taking into account the number of singletons, the estimated RTI was 86%. This study had several limitations: the first was the use of spoligotyping for genotyping of the isolates, which may have led to an overestimation of clustering compared to the other molecular techniques. This technique is also unable to differentiate between mixed infections, which might contribute to
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clustering. The second limitation is that epidemiological links between the clustered cases were not confirmed. As a result, the high percentage of clustering in this study does not provide absolute evidence of recent transmission, rather indicating potential for recent transmission. The most common spoligotype family identified by the present study was T; the predominant lineages were Euro-American and Indo-Oceanic. In line with previous studies in Ethiopia, our study findings showed that the T and CAS genotypes were the dominant families.21,25 The Euro-American lineage might have been introduced to Ethiopia by Europeans during the Italian invasion of Ethiopia, while the Indo-Oceanic lineage may have originated in Ethiopia and may thus be the ancestor of other isolates in the country. The identification of a significant proportion (13.1%) of new strains of M. tuberculosis may suggest geographic localisation of these strains in Ethiopia. Further studies using more powerful molecular tools are required to generate more information on MDR-TB in Ethiopia. As TB is increasingly becoming a health burden in the country,6 reinforcement of existing control measures is required.
CONCLUSION A large proportion of our MDR-TB isolates belonged to clusters, potentially indicating recent transmission of MDR-TB isolates in the community. Many strains that were new to SpolDB4 were identified, which could suggest the geographic localisation of these strains in the country. Surveillance and monitoring of MDR-TB strains would be useful to enhance TB control in Ethopia. Acknowledgements The authors thank the Akililu Lemma Institute of Pathobiology for financial support of this study, and the staff at St Peter TB Specialised Hospital for sourcing the multidrug-resistant tuberculosis isolates. Conflict of interest: none declared.
References 1 World Health Organization. Global tuberculosis control: WHO report. WHO/HTM/TB/2010.7. Geneva, Switzerland: WHO, 2010. 2 Federal Ministry of Health, Tuberculosis Prevention and Control Programme. Annual bulletin. Addis Ababa, Ethiopia: FMOH, 2011. 3 World Health Organization. Multidrug and extensively drugresistant TB (M/XDR-TB): 2010 global report on surveillance and response. WHO/HTM/TB/2010.3. Geneva, Switzerland: WHO, 2010. 4 Raviglione M C, Smith I M. XDR tuberculosis—implications for global public health. N Engl J Med 2007; 356: 656–659. 5 Weyer K. The management of multidrug-resistant tuberculosis in South Africa. 2nd ed. Pretoria, South Africa: Department of Health, Republic of South Africa, 1999. 6 World Health Organization. Global tuberculosis control: surveillance, planning and financing. WHO report. WHO/HTM/ TB/2008.393. Geneva, Switzerland: WHO, 2008.
7 Federal Ministry of Health. Guide line for programme and clinical management of drug resistant tuberculosis. 1st ed. Addis Ababa, Ethiopia: FMOH, 2009. 8 World Health Organization. Towards universal access to diagnosis and treatment of multidrug-resistant and extensively drugresistant tuberculosis by 2015: WHO progress report 2011. WHO/HTM/TB/2011.3. Geneva, Switzerland: WHO, 2011. 9 Van Soolingen D. Utility of molecular epidemiology of tuberculosis. Eur Respir J 1998; 11: 795–797. 10 Kamerbeek J L, Schouls A, Kolk M, et al. Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology. J Clin Microbiol 1997; 35: 907–914. 11 Goguet de la Salmoniere Y O, Li H M, Torrea G, Bunschoten A, van Embden J, Gicquel B. Evaluation of spoligotyping in a study of the transmission of Mycobacterium tuberculosis. J Clin Microbiol 1997; 35: 2210–2214. 12 Goyal M, Saunders N A, van Embden J D A, Young D B, Shaw R J. Differentiation of Mycobacterium tuberculosis isolates by spoligotyping and IS6110 restriction fragment length polymorphism. J Clin Microbiol 1997; 35: 647–651. 13 Diaz R, Kremer K, de Haas P E W, et al. Molecular epidemiology of tuberculosis in Cuba outside of Havana, July 1994–June 1995: utility of spoligotyping versus IS6110 restriction fragment length polymorphism. Int J Tuberc Lung Dis 1998; 2: 743–750. 14 Horgen L, Sola C, Devallois A, Goh K S, Rastogi N. Follow-up of Mycobacterium tuberculosis transmission in the French West Indies by IS6110-DNA fingerprinting and DR-based spoligotyping. FEMS Immunol Med Microbiol 1998; 21: 203–210. 15 Sola C, Horgen L, Devallois A, Rastogi N. Combined numerical analysis based on the molecular description of Mycobacterium tuberculosis by four repetitive sequence-based DNA typing systems. Res Microbiol 1998; 149: 349–360. 16 Haddad N, Masselot M, Durand B: Molecular differentiation of Mycobacterium bovis isolates. Review of main techniques and applications. Res Vet Sci 2004; 76: 1–18. 17 Parsons M L, Brosch R, Stewart T, et al. Rapid and simple approach for identification of Mycobacterium tuberculosis complex isolates by PCR-based genomic deletion analysis. J Clin Microbiol 2002; 40: 2339–2345. 18 Small P M, Hopewell P C, Singh S P. The epidemiology of tuberculosis in San Francisco. A population-based study using conventional and molecular methods. N Eng J Med 1994; 330: 1703–1709. 19 Luciani F, Francis R A, Tanaka M M. Interpreting genotype cluster sizes of Mycobacterium tuberculosis isolates typed with IS6110 and spoligotyping. Infect Genet Evol 2008; 8: 182–190. 20 Sepkowitz K A, Friedman C R, Hafner A, et al. Tuberculosis among urban health care workers: a study using restriction fragment length polymorphism typing. Clin Infect Dis 1995; 21: 1098–1101. 21 Agonafir M, Lemma E, Wolde-Meskel D, et al. Phenotypic and genotypic analysis of multidrug-resistant tuberculosis in Ethiopia. Int J Tuberc Lung Dis 2010; 14: 1259–1265. 22 Brudey K, Driscoll R J, Rigouts L, et al. Mycobacterium tuberculosis complex genetic diversity: mining the fourth international spoligotyping database (SpolDB4) for classification, population genetics and epidemiology. BMC Microbiol 2006; 6: 23. 23 Gagneuxa S, DeRiemer K, Van T, et al. Variable host–pathogen compatibility in Mycobacterium tuberculosis. J Microbiol 2006; 103: 2869–2873. 24 Grassberger P. Critical behavior of the general epidemic process and dynamical percolation. Math Biosci 1983; 63: 157–172. 25 Bruchfeld J, Aderaye G, Palme I B, et al. Molecular epidemiology and drug resistance of Mycobacterium tuberculosis isolates from Ethiopian pulmonary tuberculosis patients with and without human immunodeficiency virus infection. J Clin Microbiol 2002; 1636–1643.
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RÉSUMÉ C O N T E X T E : L’Hôpital St Peter Spécialisé en Tuberculose (TB) et l’Institut de Pathologie Aklilu Lemma, Addis Abeba, Ethiopie. O B J E C T I F : Génotypage de TB multirésistant (TB-MDR) et évaluation de l’importance de son regroupement en grappes. S C H É M A : On a caractérisé au moyen d’un typage moléculaire au total 183 isolats consécutifs TB-MDR entre septembre 2009 et février 2012. Avant cette étude, le caractère TB-MDR des isolats a été confirmé au moyen du test GenoType® MTBDRplus. Pour l’analyse des grappes, on a utilisé un index de transmission récente. R É S U LTAT S : Le spoligotypage a identifié 43 types différents, parmi lesquels 17 étaient formés par au moins
deux isolats en grappe alors que 26 ne comportaient qu’un seul isolat. Les types les plus fréquents ont été le nombre de types de spoligotypage internationaux (SIT) 21 et 149. Vingt-quatre types ne correspondaient pas aux types existant dans la base de données SpolDB4. Les souches appartenaient à trois lignages, les lignages principaux étant Euro-américain et Indo-océanique, correspondant chacun à 65 isolats. Une forte proportion des patients (86%) était infectée par des souches en grappe, ce qui pourrait suggérer une transmission récente de la TB-MDR dans la zone de l’étude. C O N C L U S I O N : L’observation de la formation de grappes de types de spoligotypage des isolats TB-MDR pourrait suggérer la transmission d’une souche TB-MDR et mériterait de l’attention. RESUMEN
El Hospital Saint Peter especializado en tuberculosis y el Instituto de Patología Aklilu Lemma en Addis Abeba, Etiopía. O B J E T I V O : Genotipificar las cepas de tuberculosis multidrogorresistentes (TB-MDR) y evaluar la magnitud de la agrupación en conglomerados. M É T O D O S : Se recogieron 183 aislados clínicos consecutivos de TB-MDR entre septiembre del 2009 y febrero del 2012 y se caracterizaron mediante genotipificación molecular. Como etapa previa al estudio se confirmó la TB-MDR de las cepas mediante la prueba de GenoType® MTBDRplus. Los conglomerados se analizaron según el índice de transmisión reciente. R E S U LTA D O S : Mediante la espoligotipificación se detectaron 43 tipos diferentes de Mycobacterium tuberculosis, de los cuales 17 contaban como mínimo con dos MARCO DE REFERENCIA:
aislados agrupados en conglomerados y 26 aislados eran únicos. Los tipos más frecuentes fueron los espoligotipos internacionales (SIT) número 21 y 149. Veinticuatro genotipos no correspondieron a ninguno de los tipos definidos en la base de datos SpolDB4. Las cepas pertenecían a tres linajes, con predominio del euroamericano y el indoceánico, cada uno con 65 aislados. Se observó una alta proporción de pacientes con infección causada por cepas pertenecientes a conglomerados (86%), lo cual puede indicar una transmisión reciente del TB-MDR en la zona del estudio. C O N C L U S I Ó N : La presencia de conglomerados de genotipos de TB-MDR determinados por espoligotipificación podría corresponder a la transmisión reciente de una cepa TB-MDR en la población, lo cual precisa una atención especial.
