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1.  Mycobacterium tuberculosis complex genetic diversity: mining the fourth international spoligotyping database (SpolDB4) for classification, population genetics and epidemiology 
BMC Microbiology  2006;6:23.
The Direct Repeat locus of the Mycobacterium tuberculosis complex (MTC) is a member of the CRISPR (Clustered regularly interspaced short palindromic repeats) sequences family. Spoligotyping is the widely used PCR-based reverse-hybridization blotting technique that assays the genetic diversity of this locus and is useful both for clinical laboratory, molecular epidemiology, evolutionary and population genetics. It is easy, robust, cheap, and produces highly diverse portable numerical results, as the result of the combination of (1) Unique Events Polymorphism (UEP) (2) Insertion-Sequence-mediated genetic recombination. Genetic convergence, although rare, was also previously demonstrated. Three previous international spoligotype databases had partly revealed the global and local geographical structures of MTC bacilli populations, however, there was a need for the release of a new, more representative and extended, international spoligotyping database.
The fourth international spoligotyping database, SpolDB4, describes 1939 shared-types (STs) representative of a total of 39,295 strains from 122 countries, which are tentatively classified into 62 clades/lineages using a mixed expert-based and bioinformatical approach. The SpolDB4 update adds 26 new potentially phylogeographically-specific MTC genotype families. It provides a clearer picture of the current MTC genomes diversity as well as on the relationships between the genetic attributes investigated (spoligotypes) and the infra-species classification and evolutionary history of the species. Indeed, an independent Naïve-Bayes mixture-model analysis has validated main of the previous supervised SpolDB3 classification results, confirming the usefulness of both supervised and unsupervised models as an approach to understand MTC population structure. Updated results on the epidemiological status of spoligotypes, as well as genetic prevalence maps on six main lineages are also shown. Our results suggests the existence of fine geographical genetic clines within MTC populations, that could mirror the passed and present Homo sapiens sapiens demographical and mycobacterial co-evolutionary history whose structure could be further reconstructed and modelled, thereby providing a large-scale conceptual framework of the global TB Epidemiologic Network.
Our results broaden the knowledge of the global phylogeography of the MTC complex. SpolDB4 should be a very useful tool to better define the identity of a given MTC clinical isolate, and to better analyze the links between its current spreading and previous evolutionary history. The building and mining of extended MTC polymorphic genetic databases is in progress.
PMCID: PMC1468417  PMID: 16519816
2.  Snapshot of Moving and Expanding Clones of Mycobacterium tuberculosis and Their Global Distribution Assessed by Spoligotyping in an International Study†  
Journal of Clinical Microbiology  2003;41(5):1963-1970.
The present update on the global distribution of Mycobacterium tuberculosis complex spoligotypes provides both the octal and binary descriptions of the spoligotypes for M. tuberculosis complex, including Mycobacterium bovis, from >90 countries (13,008 patterns grouped into 813 shared types containing 11,708 isolates and 1,300 orphan patterns). A number of potential indices were developed to summarize the information on the biogeographical specificity of a given shared type, as well as its geographical spreading (matching code and spreading index, respectively). To facilitate the analysis of hundreds of spoligotypes each made up of a binary succession of 43 bits of information, a number of major and minor visual rules were also defined. A total of six major rules (A to F) with the precise description of the extra missing spacers (minor rules) were used to define 36 major clades (or families) of M. tuberculosis. Some major clades identified were the East African-Indian (EAI) clade, the Beijing clade, the Haarlem clade, the Latin American and Mediterranean (LAM) clade, the Central Asian (CAS) clade, a European clade of IS6110 low banders (X; highly prevalent in the United States and United Kingdom), and a widespread yet poorly defined clade (T). When the visual rules defined above were used for an automated labeling of the 813 shared types to define nine superfamilies of strains (Mycobacterium africanum, Beijing, M. bovis, EAI, CAS, T, Haarlem, X, and LAM), 96.9% of the shared types received a label, showing the potential for automated labeling of M. tuberculosis families in well-defined phylogeographical families. Intercontinental matches of shared types among eight continents and subcontinents (Africa, North America, Central America, South America, Europe, the Middle East and Central Asia, and the Far East) are analyzed and discussed.
PMCID: PMC154710  PMID: 12734235
3.  Genetic Biodiversity of Mycobacterium tuberculosis Complex Strains from Patients with Pulmonary Tuberculosis in Cameroon 
Journal of Clinical Microbiology  2003;41(6):2547-2553.
We analyzed DNA polymorphisms in 455 Mycobacterium tuberculosis complex isolates from 455 patients to evaluate the biodiversity of tubercle bacilli in Ouest province, Cameroon. The phenotypic and genotypic identification methods gave concordant results for 99.5% of M. tuberculosis isolates (413 strains) and for 90% of Mycobacterium africanum isolates (41 strains). Mycobacterium bovis was isolated from only one patient. Analysis of regions of difference (RD4, RD9, and RD10) proved to be an accurate and rapid method of distinguishing between unusual members of the M. tuberculosis complex. Whereas M. africanum strains were the etiologic agent of tuberculosis in 56% of cases 3 decades ago, our results showed that these strains now account for just 9% of cases of tuberculosis. We identified a group of closely genetically related M. tuberculosis strains that are currently responsible for >40% of smear-positive pulmonary tuberculosis cases in this region of Cameroon. These strains shared a spoligotype lacking spacers 23, 24, and 25 and had highly related IS6110 ligation-mediated (LM) PCR patterns. They were designated the “Cameroon family.” We did not find any significant association between tuberculosis-causing species or strain families and patient characteristics (sex, age, and human immunodeficiency virus status). A comparison of the spoligotypes of the Cameroon strains with an international spoligotype database (SpolDB3) containing 11,708 patterns from >90 countries, showed that the predominant spoligotype in Cameroon was limited to West African countries (Benin, Senegal, and Ivory Coast) and to the Caribbean area.
PMCID: PMC156567  PMID: 12791879
4.  First insight into Mycobacterium tuberculosis genetic diversity in Paraguay 
BMC Microbiology  2007;7:75.
We present a picture of the biodiversity of Mycobacterium tuberculosis in Paraguay, an inland South American country harboring 5 million inhabitants with a tuberculosis notification rate of 38/100,000.
A total of 220 strains collected throughout the country in 2003 were classified by spoligotyping into 79 different patterns. Spoligopatterns of 173 strains matched 51 shared international types (SITs) already present in an updated version of SpolDB4, the global spoligotype database at Pasteur Institute, Guadeloupe. Our study contributed to the database 13 new SITs and 15 orphan spoligopatterns. Frequencies of major M. tuberculosis spoligotype lineages in our sample were as follows: Latin-American & Mediterranean (LAM) 52.3%, Haarlem 18.2%, S clade 9.5%, T superfamily 8.6%, X clade 0.9% and Beijing clade 0.5%. Concordant clustering by IS6110 restriction fragment length polymorphism (RFLP) and spoligotyping identified transmission in specific settings such as the Tacumbu jail in Asuncion and aboriginal communities in the Chaco. LAM genotypes were ubiquitous and predominated among both RFLP clusters and new patterns, suggesting ongoing transmission and adaptative evolution in Paraguay. We describe a new and successfully evolving clone of the Haarlem 3 sub-lineage, SIT2643, which is thus far restricted to Paraguay. We confirmed its clonality by RFLP and mycobacterial interspersed repetitive unit (MIRU) typing; we named it "Tacumbu" after the jail where it was found to be spreading. One-fifth of the spoligopatterns in our study are rarely or never seen outside Paraguay and one-tenth do not fit within any of the major phylogenetic clades in SpolDB4.
Lineages currently thriving in Paraguay may reflect local host-pathogen adaptation of strains introduced during past migrations from Europe.
PMCID: PMC1988809  PMID: 17686181
5.  Mycobacterium tuberculosis spoligotypes and drug susceptibility pattern of isolates from tuberculosis patients in peri-urban Kampala, Uganda 
The poor peri-urban areas of developing countries with inadequate living conditions and a high prevalence of HIV infection have been implicated in the increase of tuberculosis (TB). Presence of different lineages of Mycobacterium tuberculosis has been described in different parts of the world. This study determined the predominant strain lineages that cause TB in Rubaga division, Kampala, Uganda, and the prevalence of resistance to key anti-tuberculosis drugs in this community.
This was a cross-sectional study of newly diagnosed sputum smear-positive patients aged ≥ 18 years. A total of 344 isolates were genotyped by standard spoligotyping and the strains were compared with those in the international spoligotype database (SpolDB4). HIV testing and anti-tuberculosis drug susceptibility assays for isoniazid and rifampicin were performed and association with the most predominant spoligotypes determined.
A total of 33 clusters were obtained from 57 spoligotype patterns. According to the SpolDB4 database, 241 (70%) of the isolates were of the T2 family, while CAS1-Kili (3.5%), LAM9 (2.6%), CAS1-Delhi (2.6%) were the other significant spoligotypes. Furthermore, a major spoligotype pattern of 17 (4.5%) strains characterized by lack of spacers 15–17 and 19–43 was not identified in SpolDB4. A total of 92 (26.7%) of the patients were HIV sero-positive, 176 (51.2%) sero-negative, while 76 (22.1%) of the patients did not consent to HIV testing. Resistance to isoniazid was found in 8.1% of strains, while all 15 (4.4%) strains resistant to rifampicin were multi-drug resistant. Additionally, there was no association between any strain types in the sample with either drug resistance or HIV sero-status of the patients.
The TB epidemic in Kampala is localized, mainly caused by the T2 family of strains. Strain types were neither associated with drug resistance nor HIV sero-status.
PMCID: PMC2519071  PMID: 18662405
6.  Molecular typing of mycobacterium tuberculosis isolates circulating in Jiangsu Province, China 
BMC Infectious Diseases  2011;11:288.
Globally, China is the second place with high burden of tuberculosis (TB). To explore the characteristics of the pathogens of Mycobacterium tuberculosis (MTB) circulating in this area is helpful for understanding and controlling the spread of the strains. Recent developments in molecular biology have allowed prompt identification and tracking specific strains of MTB spreading through the population.
Spacer-oligonucleotide typing (spoligotyping) and mycobacterial interspersed repetitive units variable number tandem repeat (MIRU-VNTR) were performed in combination to yield specific genetic profiles of 260 MTB strains isolated from 30 counties of Jiangsu province in China between June and July 2010. The spoligotyping results were in comparison to the world Spoligotyping Database of Institute Pasteur de Guadeloupe (SpolDB4). Drug susceptibility test (DST) was performed on all strains by proportion method on Lowenstein-Jensen (LJ) culture media.
Based on the spoligotyping method, 246 strains displayed known patterns and 14 were absent in the database. Predominant spoligotypes belonged to the Beijing family (80.4%). By using the 24-loci VNTR typing scheme, 224 different patterns were identified, including 20 clusters and 204 unique patterns. The largest clade comprised 195 strains belonging to the Beijing family. The combination of spoligotyping and 24-loci MIRU-VNTR demonstrated maximal discriminatory power. Furthermore, we observed a significant association between Beijing family strains and drug-resistant phenotypes. The Beijing family strains presented increased risks for developing multi-drug resistant TB, with the OR (95% CI) of 11.07(1.45-84.50).
The present study demonstrated that Beijing family isolates were the most prevalent strains circulating in Jiangsu province of China. The utility of spoligotyping in combination with 24-loci MIRU-VNTR might be a useful tool for epidemiological analysis of MTB transmission.
PMCID: PMC3215657  PMID: 22026819
7.  Characterization of the genetic diversity of Mycobacterium tuberculosis in São Paulo city, Brazil 
BMC Research Notes  2011;4:269.
Tuberculosis is a major health problem in São Paulo, Brazil, which is the most populous and one of the most cosmopolitan cities in South America. To characterize the genetic diversity of Mycobacterium tuberculosis in the population of this city, the genotyping techniques of spoligotyping and MIRU were applied to 93 isolates collected in two consecutive years from 93 different tuberculosis patients residing in São Paulo city and attending the Clemente Ferreira Institute (the reference clinic for the treatment of tuberculosis).
Spoligotyping generated 53 different spoligotype patterns. Fifty-one isolates (54.8%) were grouped into 13 spoligotyping clusters. Seventy- two strains (77.4%) showed spoligotypes described in the international databases (SpolDB4, SITVIT), and 21 (22.6%) showed unidentified patterns. The most frequent spoligotype families were Latin American Mediterranean (LAM) (26 isolates), followed by the T family (24 isolates) and Haarlem (H) (11 isolates), which together accounted for 65.4% of all the isolates. These three families represent the major genotypes found in Africa, Central America, South America and Europe. Six Spoligo-International-types (designated SITs by the database) comprised 51.8% (37/72) of all the identified spoligotypes (SIT53, SIT50, SIT42, SIT60, SIT17 and SIT1). Other SITs found in this study indicated the great genetic diversity of M. tuberculosis, reflecting the remarkable ethnic diversity of São Paulo city inhabitants. The MIRU technique was more discriminatory and did not identify any genetic clusters with 100% similarity among the 93 isolates. The allelic analysis showed that MIRU loci 26, 40, 23 and 10 were the most discriminatory. When MIRU and spoligotyping techniques were combined, all isolates grouped in the 13 spoligotyping clusters were separated.
Our data indicated the genomic stability of over 50% of spoligotypes identified in São Paulo and the great genetic diversity of M. tuberculosis isolates in the remaining SITs, reflecting the large ethnic mix of the São Paulo city inhabitants. The results also indicated that in this city, M. tuberculosis isolates acquired drug resistance independently of genotype and that resistance was more dependent on the selective pressure of treatment failure and the environmental circumstances of patients.
PMCID: PMC3160979  PMID: 21801364
8.  Use of cluster-graphs from spoligotyping data to study genotype similarities and a comparison of three indices to quantify recent tuberculosis transmission among culture positive cases in French Guiana during a eight year period 
French Guiana has the highest tuberculosis (TB) burden among all French departments, with a strong increase in the TB incidence over the last few years. It is now uncertain how best to explain this incidence. The objective of this study was to compare three different methods evaluating the extent of recent TB transmission in French Guiana.
We conducted a population-based molecular epidemiology study of tuberculosis in French Guiana based on culture-positive TB strains (1996 to 2003, n = 344) to define molecular relatedness between isolates, i.e. potential transmission events. Phylogenetic relationships were inferred by comparing two methods: a "cluster-graph" method based on spoligotyping results, and a minimum spanning tree method based on both spoligotyping and variable number of tandem DNA repeats (VNTR). Furthermore, three indices attempting to reflect the extent of recent TB transmission (RTIn, RTIn-1 and TMI) were compared.
Molecular analyses showed a total amount of 120 different spoligotyping patterns and 273 clinical isolates (79.4%) that were grouped in 49 clusters. The comparison of spoligotypes from French Guiana with an international spoligotype database (SpolDB4) showed that the majority of isolates belonged to major clades of M. tuberculosis (Haarlem, 22.6%; Latin American-Mediterranean, 23.3%; and T, 32.6%). Indices designed to quantify transmission of tuberculosis gave the following values: RTIn = 0.794, RTIn-1 = 0.651, and TMI = 0.146.
Our data showed a high number of Mycobacterium tuberculosis clusters, suggesting a high level of recent TB transmission, nonetheless an estimation of transmission rate taking into account cluster size and mutation rate of genetic markers showed a low ongoing transmission rate (14.6%). Our results indicate an endemic mode of TB transmission in French Guiana, with both resurgence of old spatially restricted genotypes, and a significant importation of new TB genotypes by migration of TB infected persons from neighgouring high-incidence countries.
PMCID: PMC2375894  PMID: 18410681
9.  Mycobacterium tuberculosis Spoligotypes in Monterrey, Mexico▿  
Journal of Clinical Microbiology  2009;48(2):448-455.
Although tuberculosis is still a public health problem in Mexico, there is little information about the genetic characteristics of the isolates. In the present study, we analyzed by spoligotyping 180 Mycobacterium tuberculosis clinical isolates from the urban area of Monterrey, Mexico, including drug-susceptible and drug-resistant isolates. The spoligotype patterns were compared with those in the international SITVIT2 spoligotyping database. Four isolates presented spoligotype patterns not found in the database (orphan types); the rest were distributed among 44 spoligo international types (SITs). SIT53 (clade T1) and SIT119 (clade X1) were predominant and included 43 (23.8%) and 28 (15.5%) of the isolates, respectively. In order to determine if there was a dominant spoligotype in the group of multidrug-resistant isolates, 37 of them were analyzed by IS6110-based restriction fragment length polymorphism assays, and scarce clustering of strains with more than five bands was observed. Fourteen isolates of this multidrug-resistant group presented four bands or less and were distributed in four SITs: SIT53 (n = 8), SIT92 (n = 3), SIT70 (n = 2), and SIT3038 (n = 1). When the molecular detection of mutations in the katG and rpoB genes were analyzed in these isolates with low copy numbers of IS6110, only two isolates shared the same IS6110, spoligotyping, and mutations patterns. When the distribution of the spoligotypes was analyzed by age cohort, SIT119 was predominantly found in patients 0 to 20 years old, especially in males, accounting for up to 40% of the isolates. In contrast, SIT53 was more prevalent in older females. This analysis demonstrates the variability of M. tuberculosis isolates in Monterrey and the partial dominance of SIT53 and SIT119 in that area of Mexico.
PMCID: PMC2815641  PMID: 19940048
10.  Molecular characterisation of Mycobacterium tuberculosis isolates in the First National Survey of Anti-tuberculosis Drug Resistance from Venezuela 
BMC Microbiology  2006;6:90.
Molecular typing of Mycobacterium tuberculosis strains has become a valuable tool in the epidemiology of tuberculosis (TB) by allowing detection of outbreaks, tracking of epidemics, identification of genotypes and transmission events among patients who would have remained undetected by conventional contact investigation. This is the first genetic biodiversity study of M. tuberculosis in Venezuela. Thus, we investigated the genetic patterns of strains isolated in the first survey of anti-tuberculosis drug-resistance realised as part of the Global Project of Anti-tuberculosis Drug Resistance Surveillance (WHO/IUATLD).
Clinical isolates (670/873) were genotyped by spoligotyping. The results were compared with the international spoligotyping database (SpolDB4). Multidrug resistant (MDR) strains (14/18) were also analysed by IS6110-RFLP assays, and resistance to isoniazid and rifampicin was characterised.
Spoligotyping grouped 82% (548/670) of the strains into 59 clusters. Twenty new spoligotypes (SITs) specific to Venezuela were identified. Eight new inter-regional clusters were created. The Beijing genotype was not found. The genetic network shows that the Latin American and Mediterranean family constitutes the backbone of the genetic TB population-structure in Venezuela, responsible of >60% of total TB cases studied. MDR was 0.5% in never treated patients and 13.5% in previously treated patients. Mutations in rpoB gene and katG genes were detected in 64% and 43% of the MDR strains, respectively.
Two clusters were found to be identical by the four different analysis methods, presumably representing cases of recent transmission of MDR tuberculosis.
This study gives a first overview of the M. tuberculosis strains circulating in Venezuela during the first survey of anti-tuberculosis drug-resistance. It may aid in the creation of a national database that will be a valuable support for further studies.
PMCID: PMC1621067  PMID: 17032442
11.  Molecular epidemiology and evolutionary genetics of Mycobacterium tuberculosis in Taipei 
The control of tuberculosis in densely populated cities is complicated by close human-to-human contacts and potential transmission of pathogens from multiple sources. We conducted a molecular epidemiologic analysis of 356 Mycobacterium tuberculosis (MTB) isolates from patients presenting pulmonary tuberculosis in metropolitan Taipei. Classical antibiogram studies and genetic characterization, using mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing and spoligotyping, were applied after culture.
A total of 356 isolates were genotyped by standard spoligotyping and the strains were compared with in the international spoligotyping database (SpolDB4). All isolates were also categorized using the 15 loci MIRU-VNTR typing method and combin with NTF locus and RD deletion analyses.
Of 356 isolates spoligotyped, 290 (81.4%) displayed known spoligotypes and 66 were not identified in the database. Major spoligotypes found were Beijing lineages (52.5%), followed by Haarlem lineages (13.5%) and EAI plus EAI-like lineages (11%). When MIRU-VNTR was employed, 140 patterns were identified, including 36 clusters by 252 isolates and 104 unique patterns, and the largest cluster comprised 95 isolates from the Beijing family. The combination of spoligotyping and MIRU-VNTR revealed that 236 (67%) of the 356 isolates were clustered in 43 genotypes. Strains of the Beijing family was more likely to be of modern strain and a higher percentage of multiple drug resistance than other families combined (P = 0.08). Patients infected with Beijing strains were younger than those with other strains (mean 58.7 vs. 64.2, p = 0.02). Moreover, 85.3% of infected persons younger than 25 years had Beijing modern strain, suggesting a possible recent spread in the young population by this family of TB strain in Taipei.
Our data on MTB genotype in Taipei suggest that MTB infection has not been optimally controlled. Control efforts should be reinforced in view of the high prevalence of the Beijing strain in young population and association with drug resistance.
PMCID: PMC2628671  PMID: 19102768
12.  First Molecular Epidemiology Study of Mycobacterium tuberculosis in Burkina Faso▿  
Journal of Clinical Microbiology  2007;45(3):921-927.
We conducted a molecular epidemiology study on 120 Mycobacterium tuberculosis isolates from patients presenting pulmonary tuberculosis (TB) in Burkina Faso. Classical antibiogram studies and genetic characterization, using mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing and spoligotyping, were applied after culture. Molecular analysis of specific signatures showed that all TB cases reported in this study were caused by M. tuberculosis and identified no Mycobacterium bovis or Mycobacterium africanum isolates. This result is unexpected, as M. africanum strains were reportedly the etiologic agent in 20% of TB cases 2 decades ago. The comparison of spoligotypes from Burkina Faso with an international spoligotype database (SpolDB4) showed that the majority of isolates belong to major clades of M. tuberculosis (Haarlem, 9%; Latin American-Mediterranean, 30%; and T, 20%). The predominant group of isolates (30%) corresponds to spoligotype 61, described in Cameroon as the “Cameroon family.” In Burkina Faso, as in Cameroon, this family could be associated with recent transmission of TB, suggesting a recent expansion in West Africa. Our data suggest a low level of primary drug resistance that may be a positive result of the Directly Observed Therapy Shortcourse program. Besides, based on spoligotyping plus MIRU-VNTR, data showed a high number of clusters in our sample, suggesting a high level of recent TB transmission in Burkina Faso. Nevertheless, an important genetic polymorphism was observed in this country, reflecting an endemicity situation where the control of TB would have less impact in the main towns.
PMCID: PMC1829100  PMID: 17251410
13.  Genotyping of Mycobacterium tuberculosis clinical isolates in two cities of Turkey: Description of a new family of genotypes that is phylogeographically specific for Asia Minor 
BMC Microbiology  2005;5:44.
Population-based bacterial genetics using repeated DNA loci is an efficient approach to study the biodiversity and phylogeographical structure of human pathogens, such as Mycobacterium tuberculosis, the agent of tuberculosis. Indeed large genetic diversity databases are available for this pathogen and are regularly updated. No population-based polymorphism data were yet available for M. tuberculosis in Turkey, at the crossroads of Eurasia.
A total of 245 DNAs from Mycobacterium tuberculosis clinical isolates from tuberculosis patients residing in Turkey (Malatya n = 147 or Ankara n = 98) were genotyped by spoligotyping, a high-throughput genotyping method based on the polymorphism of the Direct Repeat locus. Thirty-three spoligotyping-defined clusters including 206 patients and 39 unique patterns were found. The ST41 cluster, as designated according to the international SpolDB3 database project, represented one fourth and when gathered to three genotypes, ST53, ST50 and ST284, one half of all the isolates. Out of 34 clinical isolates harboring ST41 which were further genotyped by IS6110 and by MIRU-VNTR typing, a typical 2-copy IS6110-RFLP pattern and a "215125113322" MIRU-VNTR pattern were observed among 21 clinical isolates. Further search in various databases confirms the likely Turkish-phylogeographical specificity of this clonal complex.
We described a new phylogeographically-specific clone of M. tuberculosis, designated LAM7-TUR. Further investigations to assess its frequency within all regions of Turkey and its phylogeographical origin and phylogenetic position within the global M. tuberculosis phylogenetic tree will shed new light on its endemicity in Asia Minor.
PMCID: PMC1192800  PMID: 16045794
14.  Spoligotypes of Mycobacterium tuberculosis from Different Provinces of China▿ †  
Journal of Clinical Microbiology  2010;48(11):4102-4106.
A total of 2,346 Mycobacterium tuberculosis isolates from 13 provinces in China were genotyped by spoligotyping. Two hundred seventy-eight spoligotypes were identified: 2,153 isolates were grouped into 85 clusters, and the remaining 193 isolates were orphans. Comparison with the SpolDB4.0 database revealed that 118 spoligotypes had shared international type numbers in the database and the other 160 were novel. These 160 novel spoligotypes were assigned to families and subfamilies using the SpotClust program. The most prevalent family was the Beijing family (74.08%), followed by the T family (14.11%). CAS family strains were found only in the Xinjiang and Tibet regions, while EAI family strains were found only in Fujian Province. In conclusion, the present study of the M. tuberculosis population in China demonstrated that Beijing family isolates are the most prevalent strains in China and that they exhibit geographical variation. Furthermore, many new spoligotypes were found in this study.
PMCID: PMC3020837  PMID: 20739484
15.  Molecular diversity of Mycobacterium tuberculosis isolates from patients with tuberculosis in Honduras 
BMC Microbiology  2010;10:208.
Tuberculosis persists as a public health problem in Honduras. A better knowledge of the molecular characteristics of Mycobacterium tuberculosis strains will contribute to understand the transmission dynamics of the disease within the country. The aim of this study was to provide an insight of the genetic biodiversity of M. tuberculosis clinical isolates collected in Honduras between 1994 and 2002. Genotyping was performed using spoligotyping and RFLP. The spoligotypes obtained were compared with the SITVIT2 proprietary database of the Pasteur Institute of Guadeloupe.
Spoligotyping grouped 84% of the isolates into 27 clusters (2 to 43 strains per cluster). Of the 44 shared international types (SITs) identified among the Honduran stains, 8 SITs were newly identified either within the present study or after match with an orphan type previously identified in the SITVIT2 database. In addition, 16 patterns corresponded to orphan, previously unreported isolates.
The Latin American Mediterranean (LAM) lineage was the most common in this study; 55% of the strains belonged to this family. Other genotypes found were Haarlem (16%), T (16%), X-clade (6%), Unknown signature (5%) and S (1%). Only one Beijing strain was identified (0.5%).
We observed a high degree of diversity after characterizing the 43 isolates belonging to the main spoligotyping cluster (SIT 33, LAM3) with IS6110-RFLP. A total of 35 different RFLP-fingerprints were detected, of which 6 patterns corresponded to the same number of clusters comprising 14 strains.
The findings obtained in this study show that tuberculosis transmission in Honduras is due to modern M. tuberculosis lineages with high level of biodiversity.
PMCID: PMC2923133  PMID: 20678242
16.  High Prevalence of Shared International Type 53 among Mycobacterium tuberculosis Complex Strains in Retreated Patients from Côte d’Ivoire 
PLoS ONE  2012;7(9):e45363.
Genotyping methods are useful tools to provide information on tuberculosis epidemic. They can allow a better response from health authorities and the implementation of measures for tuberculosis control. This study aimed to identify the main lineages and clades of Mycobacterium tuberculosis complex strains circulating in Côte d’Ivoire.
Methods/Main Findings
Strains isolated from sputum samples of patients ongoing retreatment from all the country were characterized by spoligotyping and by MIRU-VNTR. Profiles obtained by spoligotyping were first compared to the SITVIT/SpolDB4 database for family assignment. Of 194 strains analysed, 146 (75.3%) belonged to the T lineage. The most predominant spoligotype was the shared international type 53 with 135 strains (69.6%). In contrast with neighbouring countries, LAM (11 strains, 5.7%) and H (9 strains 4.6%) lineages were slightly represented. Only 3 Beijing strains (1.5%) and 4 strains of Mycobacterium africanum (2%) were found. Analysis of the results obtained with MIRU-VNTR revealed also a high level of clustering.
The population of Mycobacterium tuberculosis complex strains among retreatment cases in Côte d’Ivoire exhibits a low diversity, allowing to assume recent transmission and locally based infection.
PMCID: PMC3445461  PMID: 23028962
17.  Population-Based Molecular Epidemiological Study of Tuberculosis in Malatya, Turkey▿  
Journal of Clinical Microbiology  2007;45(12):4027-4035.
This investigation describes drug resistance patterns and genotyping data on a total of 145 Mycobacterium tuberculosis strains isolated between 2000 and 2004 in Malatya, Turkey. Drug susceptibility results indicated a total of 20% resistant and 4.8% of multidrug-resistant isolates. Spoligotyping resulted in 25 unique patterns and 120 strains in 19 clusters (2 to 33 strains per cluster). When the results were compared to an international spoligotyping database, 19 of 25 unique patterns matched existing shared spoligotype international types (SITs). This led to the description of 38 SITs with 139 strains and 6 orphan patterns (not previously reported). Five of the SITs (SIT759, SIT1936, SIT1937, SIT1938, and SIT2285) were newly created. The most prevalent spoligotype was SIT41 (LAM7-TUR) with 33 (23.9%) isolates. The repartition of strains according to major M. tuberculosis clades (in decreasing order) was as follows: ill-defined T clade (45.7%) > Latin American and Mediterranean (LAM; 29%) > Haarlem (15.9%). Strains belonging to Central Asian (CAS), East-African Indian (EAI), Beijing, and Africanum clades were absent in this setting. IS6110-restriction fragment length polymorphism (RFLP) resulted in 19 clusters (52 strains), with a final clustering rate of 35.9% and a recent transmission rate of 22.8%. Typing based on mycobacterial interspersed repetitive units (MIRUs) permitted us to identify 65 patterns (23 orphan patterns and 42 patterns that matched existing MIRU international types in an updated database). The combination of the three typing methods allowed us to calculate a final clustering rate of 22% and a significantly lower transmission rate of 13.1%. The discrimination achieved by IS6110-RFLP/MIRUs was not significantly improved by adding spoligotyping results (1.4%). We conclude that our patient population is infected by diverse M. tuberculosis populations; however, the majority of the ongoing transmission is due to “evolutionary recent” tuberculosis lineages belonging to principal genetic group 2 (PGG2; Haarlem and LAM) and PGG3 (ill-defined T clade), and most of it is attributable to the LAM7-TUR sublineage with an enhanced phylogeographical specificity for Turkey. An absence of lineages belonging to PGG1 clones (EAI, CAS, and Beijing, essentially found in Central, South, and Southeast Asia), is noteworthy.
PMCID: PMC2168580  PMID: 17928426
18.  Molecular diversity of Mycobacterium tuberculosis strains indifferent provinces of Iran 
Iranian Journal of Microbiology  2013;5(4):366-373.
Background and Objectives
Molecular epidemiology tools are widely used in determining epidemiology of tuberculosis. Spoligotyping is a molecular epidemiology method that is used for characterization and typing of Mycobacterium tuberculosis complex strains. The method is based on polymorphism of the chromosomal DR locus consisting of identical 36-bp DRs alternating with 35-41 unique spacers. The objective of this study was to investigate the prevalence of M. tuberculosis spoligotypes in different provinces of Iran.
Materials and Methods
M. tuberculosis strains were isolated from TB patients of Mycobacteriology Research center (MRC). DNA was extracted from patient’s clinical samples. PCR was performed by using of specific primers for DR region. The amplified DNA was hybridized to the spoligotyping Membrane. Hybridized DNA was detected with ECL detection kit and by exposing ECL Hyperfilm to the membrane. The obtained result was entered to a binary format and was analyzed using SpolDB4 database.
Spoligotyping resulted in 136 different patterns. Out of 1242 M. tuberculosis strains, 1165 strains (93.8%) were classified into 59 clusters and the remaining strains (6.2 %) were singleton.
The results of present study showed that strains of CAS family were more prevalent than other strains in Iran. Other prevalent families were Haarlem, T and Beijing, respectively.
PMCID: PMC4385162  PMID: 25848506
Molecular epidemiology; Tuberculosis; Spoligotyping
19.  Molecular Diversity of Drug Resistant Mycobacterium Tuberculosis Strains in Western Turkey 
Balkan Medical Journal  2012;29(2):160-165.
The aim of this study was to investigate the molecular diversity and clonal relationship of drug resistant Mycobacterium tuberculosis strains isolated in Western Turkey.
Materials and Methods:
A total of 87 strains isolated between 2006 and 2009, eight of which were rifampicin monoresistant and 79 were multidrug resistant, were analyzed with IS6110 RFLP and spoligotyping methods.
The results of spoligotyping showed that 7% of the strains were orphans, and 8% were undefined for family in the SpolDB4 database. Major families of the strains were LAM (38%), T (35%), Haarlem (7%), Beijing (2%), S (2%) and U (1%) families. The clustering rate by spoligotyping was calculated as 75%. The most predominant SIT cluster was SIT41 (29%). According to the results of IS6110 RFLP, 71 different patterns of IS6110 were observed. Low copy number was found in 26% of the strains. When the results of two methods were combined, the final clustering rate was calculated as 26%.
The genotypical distribution of drug resistant tuberculosis isolates in our region indicates genetic diversity and the clustering rate was found low in our region. However, more comprehensive and long-term molecular epidemiological studies are needed to control the drug resistant strains.
PMCID: PMC4115856  PMID: 25206987
Mycobacterium tuberculosis; drug resistance; genotyping; epidemiology
20.  Using affinity propagation for identifying subspecies among clonal organisms: lessons from M. tuberculosis 
BMC Bioinformatics  2011;12:224.
Classification and naming is a key step in the analysis, understanding and adequate management of living organisms. However, where to set limits between groups can be puzzling especially in clonal organisms. Within the Mycobacterium tuberculosis complex (MTC), the etiological agent of tuberculosis (TB), experts have first identified several groups according to their pattern at repetitive sequences, especially at the CRISPR locus (spoligotyping), and to their epidemiological relevance. Most groups such as "Beijing" found good support when tested with other loci. However, other groups such as T family and T1 subfamily (belonging to the "Euro-American" lineage) correspond to non-monophyletic groups and still need to be refined. Here, we propose to use a method called Affinity Propagation that has been successfully used in image categorization to identify relevant patterns at the CRISPR locus in MTC.
To adequately infer the relative divergence time between strains, we used a distance method inspired by the recent evolutionary model by Reyes et al. We first confirm that this method performs better than the Jaccard index commonly used to compare spoligotype patterns. Second, we document the support of each spoligotype family among the previous classification using affinity propagation on the international spoligotyping database SpolDB4. This allowed us to propose a consensus assignation for all SpolDB4 spoligotypes. Third, we propose new signatures to subclassify the T family.
Altogether, this study shows how the new clustering algorithm Affinity Propagation can help building or refining clonal organims classifications. It also describes well-supported families and subfamilies among M. tuberculosis complex, especially inside the modern "Euro-American" lineage.
PMCID: PMC3126747  PMID: 21635750
asexual organisms; species delineation; epidemiology; DR locus; Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)
21.  Spoligotype-Based Comparative Population Structure Analysis of Multidrug-Resistant and Isoniazid-Monoresistant Mycobacterium tuberculosis Complex Clinical Isolates in Poland▿  
Journal of Clinical Microbiology  2010;48(11):3899-3909.
The spoligotyping-based population structure of multidrug-resistant (MDR) Mycobacterium tuberculosis strains isolated in Poland (n = 46), representing all culture-positive MDR tuberculosis (MDR-TB) cases, was compared to that of isoniazid (INH)-monoresistant strains (n = 71) isolated in 2004. The latter data set from a previous study (E. Augustynowicz-Kopeć, T. Jagielski, and Z. Zwolska, J. Clin. Microbiol. 2008, 46:4041-4044) represented 87% of all INH-monoresistant strains. The clustering rates and genotypic-diversity indexes for the 2 subpopulations were not significantly different (P = 0.05). The results were entered in the SITVIT2 database to assign specific shared type designations, corresponding genotypic lineages, and geographical distributions and compared to available data from neighboring countries (Germany, n = 704; Czech Republic, n = 530; Sweden, n = 379; Kaliningrad, Russia, n = 90) and strains from previous studies in Poland (n = 317). MDR strains resulted in 27 patterns (20 unique strains within the study and 7 clusters containing 2 to 6 isolates per cluster with a clustering rate of 56.5%) and belonged to the following genotypic lineages: ill-defined T family (28.3%), Haarlem (17.4%), Latin American and Mediterranean (LAM) (13%), Beijing (8.7%), S family (4.35%), and the X clade (2.17%). Comparison of the genetic structure of the MDR strains with that of INH-monoresistant strains showed that a total of 9 patterns were shared by both groups; these represented 1/3 of the MDR strains and 2/3 of the INH-monoresistant strains. Interestingly, 76.1% of the MDR isolates and 71.8% of the INH-resistant isolates yielded spoligotypes that were previously reported from Poland. The observation that nearly half of the spoligotypes identified among both MDR (48.1%) and INH-monoresistant (43.3%) M. tuberculosis isolates were present in Poland's neighboring countries suggested that a significant proportion of MDR and INH-resistant TB cases in Poland were caused by strains actively circulating in Poland or its neighbors. Our results corroborate the leading role of the T and Haarlem genotypes in the epidemiology of drug-resistant TB in Poland. Nevertheless, the LAM and Beijing family strains that infected, correspondingly, 13% and 9% of patients with MDR-TB were absent among the strains from patients with INH-monoresistant TB, suggesting that a proportion of MDR-TB cases in Poland are due to ongoing transmission of MDR clones exhibiting specific genotypes. Study of the population genetic relationships between MDR and INH-monoresistant strains by drawing minimum spanning trees showed that ill-defined T1 sublineage strains (1/3 of all INH-monoresistant strains), represented by its prototype, SIT53, constituted the central node of the tree, followed by strains belonging to the well-defined H3, H1, and S subgroups. However, the MDR group, in addition, contained LAM (n = 6) and Beijing (n = 4) lineage isolates. With the exception of the 4 Beijing lineage strains in the latter group and a single orphan isolate in the INH-monoresistant group, none of the remaining 112/117 isolates belonged to principal genetic group 1 (PGG1) in our study. Given the high rate of clustering and the near absence of immigrants in the study, the persistence of MDR-TB in Poland seems to result from active transmission of MDR strains within the autochthonous population, the bulk of it caused by evolutionarily recent tubercle bacilli.
PMCID: PMC3020827  PMID: 20810763
22.  Study of Mycobacterium tuberculosis Complex Genotypic Diversity in Malaysia Reveals a Predominance of Ancestral East-African-Indian Lineage with a Malaysia-Specific Signature 
PLoS ONE  2014;9(12):e114832.
Tuberculosis (TB) still constitutes a major public health problem in Malaysia. The identification and genotyping based characterization of Mycobacterium tuberculosis complex (MTBC) isolates causing the disease is important to determine the effectiveness of the control and surveillance programs.
This study intended a first assessment of spoligotyping-based MTBC genotypic diversity in Malaysia followed by a comparison of strains with those prevailing in neighboring countries by comparison with an international MTBC genotyping database.
Spoligotyping was performed on a total of 220 M. tuberculosis clinical isolates collected in Kelantan and Kuala Lumpur. The results were compared with the SITVIT2 international database of the Pasteur Institute of Guadeloupe.
Spoligotyping revealed 77 different patterns: 22 corresponded to orphan patterns while 55 patterns containing 198 isolates were assigned a Spoligo International Type (SIT) designation in the database (the latter included 6 newly created SITs). The eight most common SITs grouped 141 isolates (5 to 56 strains per cluster) as follows: SIT1/Beijing, n = 56, 25.5%; SIT745/EAI1-SOM, n = 33, 15.0%; SIT591/EAI6-BGD1, n = 13, 5.9%; SIT256/EAI5, n = 12, 5.5%; SIT236/EAI5, n = 10, 4.6%; SIT19/EAI2-Manila, n = 9, 4.1%; SIT89/EAI2-Nonthaburi, n = 5, 2.3%; and SIT50/H3, n = 3, 1.4%. The association between city of isolation and lineages was statistically significant; Haarlem and T lineages being higher in Kuala Lumpur (p<0.01). However, no statistically significant differences were noted when comparing drug resistance vs. major lineages, nor between gender and clades.
The ancestral East-African-Indian (EAI) lineage was most predominant followed by the Beijing lineage. A comparison of strains with those prevailing in neighboring countries in South Asia, East Asia and South East Asia underlined the phylogeographical specificity of SIT745 for Malaysia, and its probable ongoing evolution with locally evolved strains sharing a specific signature characterized by absence of spacers 37, 38, and 40. Pending complementary genotyping confirmation, we propose that SIT745/EAI-SOM is tentatively reclassified as SIT745/EAI-MYS.
PMCID: PMC4263714  PMID: 25502956
23.  Molecular Characterization of Mycobacterium tuberculosis Isolates from Different Regions of Bulgaria▿  
Journal of Clinical Microbiology  2008;46(3):1014-1018.
Mycobacterium tuberculosis isolates from different regions of Bulgaria were studied by a variety of molecular typing tools. Based on spacer oligonucleotide typing (spoligotyping), the 113 strains were subdivided into 35 spoligotypes: 5 unique profiles and 15 profiles shared by two to 29 strains; the Hunter-Gaston diversity index (HGI) was 0.9. Comparison with the international database SITVIT2 at the Institut Pasteur de Guadeloupe showed the presence of two globally distributed shared types, ST53 (25.7%) and ST47 (6.2%). Nineteen (16.8%) and six (5.3%) strains belonged to the ST125 (LAM/S subfamily) and ST41 (LAM7_TUR subfamily) types described in SITVIT2 as ubiquitous/rare and ubiquitous/common types, respectively. Seven spoligoprofiles (12 strains) were not found in the database; two of them constituted new shared types. The Beijing genotype strains were not found in the studied collection in spite of close contacts with Russia in the recent and historical past. Additional subtyping by IS6110-restriction fragment length polymorphism (RFLP) and 12-locus mycobacterial interspersed repetitive unit (MIRU)-variable number of tandem repeat analyses were performed within selected spoligotypes. In particular, MIRU typing showed better discrimination within ST125 than IS6110-RFLP typing (HGI = 0.83 versus 0.39). A high gradient for ST125 in Bulgaria compared to its negligible presence in the global database and neighboring countries leads us to suggest a Bulgarian phylogeographic specificity of this spoligotype. To conclude, this first study of the Bulgarian M. tuberculosis population demonstrated its heterogeneity and predominance of several worldwide-distributed and Balkan-specific spoligotypes.
PMCID: PMC2268356  PMID: 18199794
24.  Analysis of Mycobacterium tuberculosis Genotypes in Madrid and Identification of Two New Families Specific to Spain-Related Settings 
Journal of Clinical Microbiology  2005;43(4):1797-1806.
In Spain, tuberculosis (TB) patterns are changing because of the recent increase in the number of cases among immigrants. To establish the composition of circulating Mycobacterium tuberculosis strains before the effects of foreign strains appear, this study focused on molecular characterization of 233 patient isolates using spoligotyping. The spoligotyping data were further analyzed using an international database, SpolDB4. The results obtained showed that the general features of the M. tuberculosis population in Spain are coherent with those of other European countries, with the Latin American and Mediterranean group, and with the Haarlem 3 and T1 families as the most prevalent genotypes. The Spanish isolates clustered mostly with genotypes which had previously been isolated in countries linked with Spain. We also describe and fully characterize two novel M. tuberculosis families, Madrid1 and Madrid2, which are specific to Spain-related settings. The data reported here provide a solid reference when monitoring changes in the composition of the M. tuberculosis population in Spain as a consequence of the increasing rate of TB in the foreign population.
PMCID: PMC1081327  PMID: 15815001
25.  Mycobacterium tuberculosis spoligotypes and drug susceptibility pattern of isolates from tuberculosis patients in South-Western Uganda 
Determination of the prevalence and drug susceptibility of the M. tuberculosis strains is important in tuberculosis control. We determined the genetic diversity and susceptibility profiles of mycobacteria isolated from tuberculosis patients in Mbarara, South Western Uganda.
We enrolled, consecutively; all newly diagnosed and previously treated smear-positive TB patients aged ≥ 18 years. The isolates were characterized using regions of difference (RD) analysis and spoligotyping. Drug resistance against rifampicin and isoniazid were tested using the Genotype® MDRTBplus assay and the indirect proportion method on Lowenstein-Jensen media. HIV-1 testing was performed using two rapid HIV tests.
A total of 125 isolates from 167 TB suspects (60% males) with a mean age 33.7 years and HIV prevalence of 67.9% (55/81) were analyzed. Majority (92.8%) were new cases while only 7.2% were retreatment cases. All the 125 isolates were identified as M. tuberculosis strict sense with the majority (92.8%) of the isolates being modern strains while seven (7.2%) isolates were ancestral strains. Spoligotyping revealed 79 spoligotype patterns, with an overall diversity of 63.2%. Sixty two (49.6%) of the isolates formed 16 clusters consisting of 2-15 isolates each. A majority (59.2%) of the isolates belong to the Uganda genotype group of strains. The major shared spoligotypes in our sample were SIT 135 (T2-Uganda) with 15 isolates and SIT 128 (T2) with 3 isolates. Sixty nine (87%) of the 79 patterns had not yet been defined in the SpolDB4.0.database. Resistance mutations to either RIF or INH were detected in 6.4% of the isolates. Multidrug resistance, INH and RIF resistance was 1.6%, 3.2% and 4.8%, respectively. The rpoβ gene mutations seen in the sample were D516V, S531L, H526Y H526D and D516V, while one strain had a Δ1 mutation in the wild type probes. There were three strains with katG (codon 315) gene mutations only while one strain showed the inhA promoter gene mutation.
The present study shows that the TB epidemic in Mbarara is caused by modern M. tuberculosis strains mainly belonging to the Uganda genotype and anti-TB drug resistance rate in the region is low.
PMCID: PMC3100262  PMID: 21453482

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