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1.  Suitability of IS6110-RFLP and MIRU-VNTR for Differentiating Spoligotyped Drug-Resistant Mycobacterium tuberculosis Clinical Isolates from Sichuan in China 
BioMed Research International  2014;2014:763204.
Genotypes of Mycobacterium tuberculosis complex (MTBC) vary with the geographic origin of the patients and can affect tuberculosis (TB) transmission. This study was aimed to further differentiate spoligotype-defined clusters of drug-resistant MTBC clinical isolates split in Beijing (n = 190) versus non-Beijing isolates (n = 84) from Sichuan region, the second high-burden province in China, by IS6110-restriction fragment length polymorphism (RFLP) and 24-locus MIRU-VNTRs. Among 274 spoligotyped isolates, the clustering ratio of Beijing family was 5.3% by 24-locus MIRU-VNTRs versus 2.1% by IS6110-RFLP, while none of the non-Beijing isolates were clustered by 24-locus MIRU-VNTRs versus 9.5% by IS6110-RFLP. Hence, neither the 24-locus MIRU-VNTR was sufficient enough to fully discriminate the Beijing family, nor the IS6110-RFLP for the non-Beijing isolates. A region adjusted scheme combining 12 highly discriminatory VNTR loci with IS6110-RFLP was a better alternative for typing Beijing strains in Sichuan than 24-locus MIRU-VNTRs alone. IS6110-RFLP was for the first time introduced to systematically genotype MTBC in Sichuan and we conclude that the region-adjusted scheme of 12 highly discriminative VNTRs might be a suitable alternative to 24-locus MIRU-VNTR scheme for non-Beijing strains, while the clusters of the Beijing isolates should be further subtyped using IS6110-RFLP for optimal discrimination.
doi:10.1155/2014/763204
PMCID: PMC3958788  PMID: 24724099
2.  Molecular Epidemiology and Genotyping of Mycobacterium tuberculosis Isolated in Baghdad 
BioMed Research International  2014;2014:580981.
Tuberculosis (TB) remains a major health problem in Iraq but the strains responsible for the epidemic have been poorly characterized. Our aim was to characterize the TB strains circulating in Bagdad (Iraq). A total of 270 Mycobacterium tuberculosis complex (MTBC) strains isolated between 2010 and 2011 from TB patients attending the Center of Chest and Respiratory diseases in Baghdad were analyzed by Spoligotyping. The analysis indicated that 94.1% of the isolates belong to known genotype clades: CAS 39.6%, ill-defined T clade 29.6%, Manu 7.4%, Haarlem 7%, Ural 4.1%, LAM 3.3%, X 0.7%, LAM7-TUR 0.7%, EAI 0.7%, S 0.7%, and unknown 5.9%. Comparison with the international multimarker database SITVIT2 showed that SIT 309 (CAS1-Delhi) and SIT1144 (T1) were the most common types. In addition, 44 strains were included in SITVIT2 database under 16 new Spoligotype International Types (SITs); of these, 6 SITs (SIT3346, SIT3497, SIT3708, SIT3790, SIT3791, and SIT3800) (n = 32 strains) were created within the present study and 10 were created after a match with an orphan in the database. By using 24-loci MIRU-VNTR-typing on a subset of 110 samples we found a high recent transmission index (RTI) of 33.6%. In conclusion, we present the first unifying framework for both epidemiology and evolutionary analysis of M. tuberculosis in Iraq.
doi:10.1155/2014/580981
PMCID: PMC3955663  PMID: 24719873
3.  Current Methods in the Molecular Typing of Mycobacterium tuberculosis and Other Mycobacteria 
BioMed Research International  2014;2014:645802.
In the epidemiology of tuberculosis (TB) and nontuberculous mycobacterial (NTM) diseases, as in all infectious diseases, the key issue is to define the source of infection and to disclose its routes of transmission and dissemination in the environment. For this to be accomplished, the ability of discerning and tracking individual Mycobacterium strains is of critical importance. Molecular typing methods have greatly improved our understanding of the biology of mycobacteria and provide powerful tools to combat the diseases caused by these pathogens. The utility of various typing methods depends on the Mycobacterium species under investigation as well as on the research question. For tuberculosis, different methods have different roles in phylogenetic analyses and person-to-person transmission studies. In NTM diseases, most investigations involve the search for environmental sources or phylogenetic relationships. Here, too, the type of setting determines which methodology is most suitable. Within this review, we summarize currently available molecular methods for strain typing of M. tuberculosis and some NTM species, most commonly associated with human disease. For the various methods, technical practicalities as well as discriminatory power and accomplishments are reviewed.
doi:10.1155/2014/645802
PMCID: PMC3914561  PMID: 24527454
4.  Molecular Epidemiology of Tuberculosis in Finland, 2008-2011 
PLoS ONE  2013;8(12):e85027.
In industrialized countries the majority of tuberculosis (TB) cases are linked to immigration. In Finland, most cases are still Finnish born but the number of foreign born cases is steadily increasing. In this 4-year population based study, the TB situation in Finland was characterized by a genotypic analysis of Mycobacterium tuberculosis isolates. A total of 1048 M. tuberculosis isolates (representing 99.4% of all culture positive cases) were analyzed by spoligotyping and MIRU. Spoligotype lineages belonging to the Euro-American family were predominant among the Finnish isolates, particularly T (n=346, 33.0%) and Haarlem (n=237, 22.6%) strains. The lineage signature was unknown for 130 (12.4%) isolates. Out of the 17 multi-drug resistant TB strains, 10 (58.8%) belonged to the Beijing lineage. In total, 23 new SIT designations were given and 51 orphan strains were found, of which 58 patterns were unique to Finland. Phylogeographical TB mapping as compared to neighboring countries showed that the population structure in Finland most closely resembled that observed in Sweden. By combining spoligotyping and MIRU results, 98 clusters comprising 355 isolates (33.9%) were found. Only 10 clusters contained both Finnish and foreign born cases. In conclusion, a large proportion of the M. tuberculosis isolates were from Finnish born elderly patients. Moreover, many previously unidentified spoligotype profiles and isolates belonging to unknown lineages were encountered.
doi:10.1371/journal.pone.0085027
PMCID: PMC3873426  PMID: 24386443
5.  Nontuberculous Mycobacteria in Guadeloupe, Martinique, and French Guiana from 1994 to 2012 
Nontuberculous mycobacteria (NTM) are ubiquitous environmental organisms able to cause severe opportunistic human infections. Their distribution patterns are subject to geographical variations. This study describes their isolation frequencies from clinical specimen in the three French overseas departments of the Americas, namely, Guadeloupe, Martinique, and French Guiana during 1994–2012. A total of 651 strains from as many patients (one isolate per species per patient) were analysed regarding regional isolation patterns and potential pattern changes over time. The Mycobacterium avium complex was the most common group of NTM in Guadeloupe and French Guiana. In Martinique it was the second most common after the rapidly growing mycobacteria. M. fortuitum was the most commonly isolated species in all three departments. Some species (M. kansasii, M. xenopi, and M. terrae complex) displayed a clear regional preference. Furthermore a change in isolation frequency was observed for M. intracellulare (increase) and M. kansasii (decrease) in Guadeloupe. In conclusion, marked regional differences in isolation frequencies of NTM species were observed in the study area. Results are discussed in context of variables such as study populations, risk factors, methodology employed, isolation from pulmonary versus sterile isolation sites (blood, urine, and CSF), and in vitro drug-susceptibility patterns.
doi:10.1155/2013/472041
PMCID: PMC3880762  PMID: 24455239
6.  Epidemic Spread of Multidrug-Resistant Tuberculosis in Johannesburg, South Africa 
Journal of Clinical Microbiology  2013;51(6):1818-1825.
Numerous reports have documented isolated transmission events or clonal outbreaks of multidrug-resistant Mycobacterium tuberculosis strains, but knowledge of their epidemic spread remains limited. In this study, we evaluated drug resistance, strain diversity, and clustering rates in patients diagnosed with multidrug-resistant (MDR) tuberculosis (TB) at the National Health Laboratory Service (NHLS) Central TB Laboratory in Johannesburg, South Africa, between March 2004 and December 2007. Phenotypic drug susceptibility testing was done using the indirect proportion method, while each isolate was genotyped using a combination of spoligotyping and 12-MIRU typing (12-locus multiple interspersed repetitive unit typing). Isolates from 434 MDR-TB patients were evaluated, of which 238 (54.8%) were resistant to four first-line drugs (isoniazid, rifampin, ethambutol, and streptomycin). Spoligotyping identified 56 different strains and 28 clusters of variable size (2 to 71 cases per cluster) with a clustering rate of 87.1%. Ten clusters included 337 (77.6%) of all cases, with strains of the Beijing genotype being most prevalent (16.4%). Combined analysis of spoligotyping and 12-MIRU typing increased the discriminatory power (Hunter Gaston discriminatory index [HGDI] = 0.962) and reduced the clustering rate to 66.8%. Resolution of Beijing genotype strains was further enhanced with the 24-MIRU-VNTR (variable-number tandem repeat) typing method by identifying 15 subclusters and 19 unique strains from twelve 12-MIRU clusters. High levels of clustering among a variety of strains suggest a true epidemic spread of MDR-TB in the study setting, emphasizing the urgency of early diagnosis and effective treatment to reduce transmission within this community.
doi:10.1128/JCM.00200-13
PMCID: PMC3716102  PMID: 23554196
7.  Population structure and circulating genotypes of drug-sensitive and drug-resistant Mycobacterium tuberculosis clinical isolates in São Paulo state, Brazil 
São Paulo is the most populous Brazilian state and reports the largest number of tuberculosis cases in the country annually (over 18,500). This study included 193 isolates obtained during the 2nd Nationwide Survey on Mycobacterium tuberculosis Drug Resistance that was conducted in São Paulo state and 547 isolates from a laboratory based study of drug resistance that were analyzed by the Mycobacteria Reference Laboratory at the Institute Adolfo Lutz. Both studies were conducted from 2006 to 2008 and sought to determine the genetic diversity and pattern of drug resistance of M. tuberculosis isolates (MTC) circulating in São Paulo. The patterns obtained from the spoligotyping analysis demonstrated that 51/740 (6.9%) of the isolates corresponded to orphan patterns and that 689 (93.1%) of the isolates distributed into 144 shared types, including 119 that matched a preexisting shared type in the SITVIT2 database and 25 that were new isolates. A total of 77/144 patterns corresponded to unique isolates, while the remaining 67 corresponded to clustered patterns (n = 612 isolates clustered into groups of 2–84 isolates each). The evolutionarily ancient PGG1 lineages (Beijing, CAS1-DEL, EAI3-IND, and PINI2) were rarely detected in São Paulo and comprised only 13/740, or 1.76%, of the total isolates; all of the remaining 727/740, or 98.24%, of the MTC isolates from São Paulo state were from the recent PGG2/3 evolutionary isolates belonging to the LAM, T, S, X, and Haarlem lineages, i.e., the Euro-American group. This study provides the first overview of circulating genotypes of M. tuberculosis in São Paulo state and demonstrates that the clustered shared types containing seven or more M. tuberculosis isolates that are spread in São Paulo state included both resistant and susceptible isolates.
doi:10.1016/j.meegid.2012.10.015
PMCID: PMC3744825  PMID: 23201043
Mycobacterium tuberculosis; Population-structure; Spoligotyping; Brazil; Database
8.  Mycobacterium tuberculosis Beijing Genotype Is Associated with HIV Infection in Mozambique 
PLoS ONE  2013;8(8):e71999.
The Beijing genotype is a lineage of Mycobacterium tuberculosis that is distributed worldwide and responsible for large epidemics, associated with multidrug-resistance. However, its distribution in Africa is less understood due to the lack of data. Our aim was to investigate the prevalence and possible transmission of Beijing strains in Mozambique by a multivariate analysis of genotypic, geographic and demographic data. A total of 543 M. tuberculosis isolates from Mozambique were spoligotyped. Of these, 33 were of the Beijing lineage. The genetic relationship between the Beijing isolates were studied by identification of genomic deletions within some Regions of Difference (RD), Restriction Fragment Length Polymorphism (RFLP) and Mycobacterial Interspersed Repetivie Unit – variable number tandem repeat (MIRU-VNTR). Beijing strains from South Africa, representing different sublineages were included as reference strains. The association between Beijing genotype, Human Immunodeficiency Virus (HIV) serology and baseline demographic data was investigated. HIV positive serostatus was significantly (p=0.023) more common in patients with Beijing strains than in patients with non-Beijing strains in a multivariable analysis adjusted for age, sex and province (14 (10.9%) of the 129 HIV positive patients had Beijing strains while 6/141 (4.3%) of HIV negative patients had Beijing strains). The majority of Beijing strains were found in the Southern region of Mozambique, particularly in Maputo City (17%). Only one Beijing strain was drug resistant (multi-drug resistant). By combined use of RD and spoligotyping, three genetic sublineages could be tentatively identified where a distinct group of four isolates had deletion of RD150, a signature of the “sublineage 7” recently emerging in South Africa. The same group was very similar to South African “sublineage 7” by RFLP and MIRU-VNTR, suggesting that this sublineage could have been recently introduced in Mozambique from South Africa, in association with HIV infection.
doi:10.1371/journal.pone.0071999
PMCID: PMC3737140  PMID: 23940801
9.  Use of genotyping based clustering to quantify recent tuberculosis transmission in Guadeloupe during a seven years period: analysis of risk factors and access to health care 
BMC Infectious Diseases  2013;13:364.
Background
The present study aimed to characterize Mycobacterium tuberculosis population structure and to identify transmission chains and risk factors by prospective molecular typing in conjunction with conventional epidemiological investigations in the French overseas department of Guadeloupe.
Methods
The study included all the culture-positive TB cases (1 clinical isolate per patient; n = 129) diagnosed between a seven year period (April 4th, 1999 to December 31st, 2005). Prospective molecular typing was performed using spoligotyping and VNTRs, and a subset of 44 M. tuberculosis isolates found to be clustered was retrospectively typed using 12-loci MIRUs. Data were compared using the SITVIT2 database, followed by analysis of risk factors in function of clustering of the isolates and available demographic and socioeconomic data.
Results
The study sample was characterized by a majority of new cases (87.4%); a moderate proportion of drug-resistance (7.8%); a high level of immigration (51.2% foreign-born) originating from high TB/HIV incidence neighboring islands such as Haiti or Dominican Republic; lower socioeconomic conditions (70.7% of jobless, average income 824 EUR/month); and a significantly higher proportion of TB/HIV co-infected cases (38.2% vs. 8.5%; p < 0.001), and extrapulmonary disease (18.2% vs. 4.8%; p < 0.02) among migrants as compared to French patients. The study revealed an important delay in access to healthcare with a median delay of 74.5 days between the 1st symptoms and clinical suspicion of TB. Prospective molecular typing based on spoligotyping and 5-loci VNTRs showed that evolutionary recent Euro-American lineages predominated in Guadeloupe (91.5% of isolates). In conjunction with epidemiological data, it allowed to estimate a recent transmission rate of 18.6%, which was close to the rate of 16.7% estimated using retrospective 12-loci MIRU typing. Although a higher proportion of cases in older age-group were apparently linked to reactivation; univariate analysis of risk factors did not allow pinpointing specific risk factors for a patient to belong to a TB transmission group.
Conclusions
Ongoing TB transmission in the insular, low TB-incidence setting of Guadeloupe can be defined as follows: (i) a significant proportion of imported cases of the disease from neighboring islands; (ii) significantly higher TB/HIV coinfection among foreign-born cases; and, (iii) a higher proportion of cases affecting older age-group among French patients due to reactivation. This study emphasizes the need for universal typing using spoligotyping and 15-loci MIRUs in prospective studies.
doi:10.1186/1471-2334-13-364
PMCID: PMC3750484  PMID: 23914829
Mycobacterium; Tuberculosis; Transmission; Guadeloupe; Genotyping; Spoligotyping; Exact-Tandem-Repeats; Database; Drug-resistance
10.  Genetic Diversity of Mycobacterium tuberculosis in Peru and Exploration of Phylogenetic Associations with Drug Resistance 
PLoS ONE  2013;8(6):e65873.
Background
There is limited available data on the strain diversity of M tuberculosis in Peru, though there may be interesting lessons to learn from a setting where multidrug resistant TB has emerged as a major problem despite an apparently well-functioning DOTS control programme.
Methods
Spoligotyping was undertaken on 794 strains of M tuberculosis collected between 1999 and 2005 from 553 community-based patients and 241 hospital-based HIV co-infected patients with pulmonary tuberculosis in Lima, Peru. Phylogenetic and epidemiologic analyses permitted identification of clusters and exploration of spoligotype associations with drug resistance.
Results
Mean patient age was 31.9 years, 63% were male and 30.4% were known to be HIV+. Rifampicin mono-resistance, isoniazid mono-resistance and multidrug resistance (MDR) were identified in 4.7%, 8.7% and 17.3% of strains respectively. Of 794 strains from 794 patients there were 149 different spoligotypes. Of these there were 27 strains (3.4%) with novel, unique orphan spoligotypes. 498 strains (62.7%) were clustered in the nine most common spoligotypes: 16.4% SIT 50 (clade H3), 12.3% SIT 53 (clade T1), 8.3% SIT 33 (LAM3), 7.4% SIT 42 (LAM9), 5.5% SIT 1 (Beijing), 3.9% SIT 47 (H1), 3.0% SIT 222 (clade unknown), 3.0% SIT1355 (LAM), and 2.8% SIT 92 (X3). Amongst HIV-negative community-based TB patients no associations were seen between drug resistance and specific spoligotypes; in contrast HIV-associated MDRTB, but not isoniazid or rifampicin mono-resistance, was associated with SIT42 and SIT53 strains.
Conclusion
Two spoligotypes were associated with MDR particularly amongst patients with HIV. The MDR-HIV association was significantly reduced after controlling for SIT42 and SIT53 status; residual confounding may explain the remaining apparent association. These data are suggestive of a prolonged, clonal, hospital-based outbreak of MDR disease amongst HIV patients but do not support a hypothesis of strain-specific propensity for the acquisition of resistance-conferring mutations.
doi:10.1371/journal.pone.0065873
PMCID: PMC3691179  PMID: 23826083
11.  Multidrug-resistant tuberculosis in Port-au-Prince, Haiti 
Objectives
Determine the prevalence of MDR-TB among patients with new smear-positive pulmonary TB in Port au Prince, Haiti
Methods
Sputum samples were cultured from 1006 patients diagnosed with new tuberculosis in 2008. The core region of the rpoB gene that is associated with resistance to rifampin was sequenced. All isolates with rpoB mutations were sent to the NY State reference laboratory for conventional drug susceptibility testing (DST). All isolates were also tested with the GenoType MTBDRplus line probe assay.
Results
M.tubercuosis was isolated from 906 patients. 26 (2.9%) of the isolates had mis-sense mutations or deletions in rpoB and were resistant to rifampin by DST. All 26 were also resistant to isoniazid and classified as MDR-TB. 46 control isolates without rpoB mutations were all found to be rifampin-sensitive by DST. The GenoType MTBDRplus line probe assay correctly identified 26 MDR-TB strains. It misclassified 1 pansusceptible isolate as RIF-resistant.
Conclusions
This study shows an MDR-TB prevalence of 2.9% in newly diagnosed TB patients in Haiti and also suggests that rpoB sequencing or hybridization assays are good screening tools for early detection of MDR-TB.
PMCID: PMC3676925  PMID: 22569696
Tuberculosis; Multidrug-Resistant; Haiti
13.  The Genotypic Population Structure of Mycobacterium tuberculosis Complex from Moroccan Patients Reveals a Predominance of Euro-American Lineages 
PLoS ONE  2012;7(10):e47113.
Background
Tuberculosis (TB) remains a major health problem in Morocco. Characterization of circulating Mycobacterium tuberculosis genotypic lineages, important to understand the dynamic of the disease, was hereby addressed for the first time at a national level.
Methodology/Principal Findings
Spoligotyping was performed on a panel of 592 M. tuberculosis complex strains covering a 2-year period (2004–2006). It identified 129 patterns: 105 (n = 568 strains) corresponded to a SIT number in the SITVIT2 database, while 24 patterns were labeled as orphan. A total of 523 (88.3%) strains were clustered vs. 69 or 11.7% unclustered. Classification of strains within 3 large phylogenetical groups was as follows: group 1– ancestral/TbD1+/PGG1 (EAI, Bovis, Africanum), group 2– modern/TbD1−/PGG1 group (Beijing, CAS), group 3– evolutionary recent/TbD1−/PGG2/3 (Haarlem, X, S, T, LAM; alternatively designated as the Euro-American lineage). As opposed to group 3 strains (namely LAM, Haarlem, and T) that predominated (86.5% of all isolates), 6 strains belonged to group 2 (Beijing n = 5, CAS n = 1), and 3 strains (BOV_1 n = 2, BOV_4-CAPRAE) belonged to ancestral group 1 (EAI and AFRI lineage strains were absent). 12-loci MIRU-VNTR typing of the Casablanca subgroup (n = 114 strains) identified 71 patterns: 48 MITs and 23 orphan patterns; it allowed to reduce the clustering rate from 72.8% to 29.8% and the recent transmission rate from 64% to 20.2%.
Conclusion
The M. tuberculosis population structure in Morocco is highly homogeneous, and is characterized by the predominance of the Euro-American lineages, namely LAM, Haarlem, and T, which belong to the “evolutionary recent” TbD1−/PGG2/3 phylogenetic group. The combination of spoligotyping and MIRUs decreased the clustering rate significantly, and should now be systematically applied in larger studies. The methods used in this study appear well suited to monitor the M. tuberculosis population structure for an enhanced TB management program in Morocco.
doi:10.1371/journal.pone.0047113
PMCID: PMC3471964  PMID: 23077552
14.  Mycobacterium tuberculosis Strains Potentially Involved in the TB Epidemic in Sweden a Century Ago 
PLoS ONE  2012;7(10):e46848.
A hundred years ago the prevalence of tuberculosis (TB) in Sweden was one of the highest in the world. In this study we conducted a population-based search for distinct strains of Mycobacterium tuberculosis complex isolated from patients born in Sweden before 1945. Many of these isolates represent the M. tuberculosis complex population that fueled the TB epidemic in Sweden during the first half of the 20th century.
Methods
Genetic relationships between strains that caused the epidemic and present day strains were studied by spoligotyping and restriction fragment length polymorphism.
Results
The majority of the isolates from the elderly population were evolutionary recent Principal Genetic Group (PGG)2/3 strains (363/409 or 88.8%), and only a low proportion were ancient PGG1 strains (24/409 or 5.9%). Twenty-two were undefined. The isolates demonstrated a population where the Euro-American superlineage dominated; in particular with Haarlem (41.1%) and T (37.7%) spoligotypes and only 21.2% belonged to other spoligotype families. Isolates from the elderly population clustered much less frequently than did isolates from a young control group population.
Conclusions
A closely knit pool of PGG2/3 strains restricted to Sweden and its immediate neighbours appears to have played a role in the epidemic, while PGG1 strains are usually linked to migrants in todaýs Sweden. Further studies of these outbreak strains may give indications of why the epidemic waned.
doi:10.1371/journal.pone.0046848
PMCID: PMC3466202  PMID: 23056484
15.  MLVA Based Classification of Mycobacterium tuberculosis Complex Lineages for a Robust Phylogeographic Snapshot of Its Worldwide Molecular Diversity 
PLoS ONE  2012;7(9):e41991.
Multiple-locus variable-number tandem repeat analysis (MLVA) is useful to establish transmission routes and sources of infections for various microorganisms including Mycobacterium tuberculosis complex (MTC). The recently released SITVITWEB database contains 12-loci Mycobacterial Interspersed Repetitive Units – Variable Number of Tandem DNA Repeats (MIRU-VNTR) profiles and spoligotype patterns for thousands of MTC strains; it uses MIRU International Types (MIT) and Spoligotype International Types (SIT) to designate clustered patterns worldwide. Considering existing doubts on the ability of spoligotyping alone to reveal exact phylogenetic relationships between MTC strains, we developed a MLVA based classification for MTC genotypic lineages. We studied 6 different subsets of MTC isolates encompassing 7793 strains worldwide. Minimum spanning trees (MST) were constructed to identify major lineages, and the most common representative located as a central node was taken as the prototype defining different phylogenetic groups. A total of 7 major lineages with their respective prototypes were identified: Indo-Oceanic/MIT57, East Asian and African Indian/MIT17, Euro American/MIT116, West African-I/MIT934, West African-II/MIT664, M. bovis/MIT49, M.canettii/MIT60. Further MST subdivision identified an additional 34 sublineage MIT prototypes. The phylogenetic relationships among the 37 newly defined MIRU-VNTR lineages were inferred using a classification algorithm based on a bayesian approach. This information was used to construct an updated phylogenetic and phylogeographic snapshot of worldwide MTC diversity studied both at the regional, sub-regional, and country level according to the United Nations specifications. We also looked for IS6110 insertional events that are known to modify the results of the spoligotyping in specific circumstances, and showed that a fair portion of convergence leading to the currently observed bias in phylogenetic classification of strains may be traced back to the presence of IS6110. These results shed new light on the evolutionary history of the pathogen in relation to the history of peopling and human migration.
doi:10.1371/journal.pone.0041991
PMCID: PMC3439451  PMID: 22984400
16.  Molecular epidemiology and genetic diversity of Mycobacterium tuberculosis complex in the Cross River State, Nigeria 
Infection, Genetics and Evolution  2011;12(4):671-677.
This study provides with a first insight on Mycobacterium tuberculosis complex epidemiology and genetic diversity in the Cross River State, Nigeria. Starting with 137 smear positive patients recruited over a period of 12 months (June 2008 to May 2009), we obtained 97 pure mycobacterial isolates out of which 81 (83.5%) were identified as M. tuberculosis complex. Genotyping revealed a total of 27 spoligotypes patterns with 10 clusters (n = 64% or 79% of clustered isolates, 2–32 isolates/cluster), with patients in the age group range 25–34 years being significantly associated with shared-type pattern SIT61 (p = 0.019). Comparison with SITVIT2 database showed that with the exception of a single cluster (SIT727/H1), all other clusters observed were representative of West Africa; the two main lineages involved were LAM10-CAM (n = 42/81% or 51.8%) of M. tuberculosis and AFRI_2 sublineage of Mycobacterium africanum (n = 27/81% or 33.3%). Subsequent 12-loci MIRU typing resulted in a total of 13 SIT/MIT clusters (n = 52 isolates, 2–9 isolates per cluster), with a resulting recent n − 1 transmission rate of 48.1%. Available drug-susceptibility testing (DST) results for 58/81 clinical isolates revealed 6/58% or 10.4% cases of multiple drug-resistance (MDR); 5/6 MDR cases were caused by strains belonging to LAM10-CAM lineage (a specific cluster SIT61/MIT266 in 4/6 cases, and an orphan spoligotype pattern in 1/6 case). Additionally, MIT266 was associated with streptomycin resistance (p = 0.016). All the six MDRTB isolates were concomitantly resistance to streptomycin and ethambutol; however, 4/6 MDR strains with identical MIRU patterns were characterized by consecutive strain numbers hence the possibility of laboratory cross contamination could not be excluded in 3/4 serial cases. The present preliminary study underlines the usefulness of spoligotyping and 12-loci MIRU–VNTRs to establish a baseline of circulating genotypic lineages of M. tuberculosis complex in Nigeria.
doi:10.1016/j.meegid.2011.08.011
PMCID: PMC3369698  PMID: 21878397
Mycobacterium tuberculosis; Epidemiology; Spoligotyping; MIRU–VNTRs; Cross River State; Nigeria
17.  Genetic characterization of Mycobacterium tuberculosis in the West Bank, Palestinian Territories 
BMC Research Notes  2012;5:270.
Background
The World Health Organization (WHO) declared human tuberculosis (TB) a global health emergency and launched the “Global Plan to Stop Tuberculosis” which aims to save a million lives by 2015. Global control of TB is increasingly dependent on rapid and accurate genetic typing of species of the Mycobacterium tuberculosis (MTB) complex including M. tuberculosis. The aim of this study was to identify and genetically characterize the MTB isolates circulating in the West Bank, Palestinian Territories. Genotyping of the MTB isolates from patients with pulmonary TB was carried out using two molecular genetic techniques, spoligotyping and mycobacterial interspersed repetitive units-variable number of tandem repeat (MIRU-VNTR) supported by analysis of the MTB specific deletion 1 (TbD1).
Findings
A total of 17 MTB patterns were obtained from the 31 clinical isolates analyzed by spoligotyping; corresponding to 2 orphans and 15 shared-types (SITs). Fourteen SITs matched a preexisting shared-type in the SITVIT2 database, whereas a single shared-type SIT3348 was newly created. The most common spoligotyping profile was SIT53 (T1 variant), identified in 35.5 % of the TB cases studied. Genetic characterization of 22 clinical isolates via the 15 loci MIRU-VNTR typing distinguished 19 patterns. The 15-loci MIT144 and MIT145 were newly created within this study. Both methods determined the present of M. bovis strains among the isolates.
Conclusions
Significant diversity among the MTB isolates circulating in the West Bank was identified with SIT53-T1 genotype being the most frequent strain. Our results are used as reference database of the strains circulating in our region and may facilitate the implementation of an efficient TB control program.
doi:10.1186/1756-0500-5-270
PMCID: PMC3441885  PMID: 22676404
Tuberculosis; Mycobacterium; Spoligotyping; MIRU; Genotyping
18.  Distribution of Spoligotyping Defined Genotypic Lineages among Drug-Resistant Mycobacterium tuberculosis Complex Clinical Isolates in Ankara, Turkey 
PLoS ONE  2012;7(1):e30331.
Background
Investigation of genetic heterogeneity and spoligotype-defined lineages of drug-resistant Mycobacterium tuberculosis clinical isolates collected during a three-year period in two university hospitals and National Tuberculosis Reference and Research Laboratory in Ankara, Turkey.
Methods and Findings
A total of 95 drug-resistant M. tuberculosis isolates collected from three different centers were included in this study. Susceptibility testing of the isolates to four major antituberculous drugs was performed using proportion method on Löwenstein–Jensen medium and BACTEC 460-TB system. All clinical isolates were typed by using spoligotyping and IS6110-restriction fragment length polymorphism (RFLP) methods. Seventy-three of the 95 (76.8%) drug resistant M. tuberculosis isolates were isoniazid-resistant, 45 (47.4%) were rifampicin-resistant, 32 (33.7%) were streptomycin-resistant and 31 (32.6%) were ethambutol-resistant. The proportion of multidrug-resistant isolates (MDR) was 42.1%. By using spoligotyping, 35 distinct patterns were observed; 75 clinical isolates were grouped in 15 clusters (clustering rate of 79%) and 20 isolates displayed unique patterns. Five of these 20 unique patterns corresponded to orphan patterns in the SITVIT2 database, while 4 shared types containing 8 isolates were newly created. The most prevalent M. tuberculosis lineages were: Haarlem (23/95, 24.2%), ill-defined T superfamily (22/95, 23.2%), the Turkey family (19/95, 20%; previously designated as LAM7-TUR), Beijing (6/95, 6.3%), and Latin-America & Mediterranean (LAM, 5/95 or 5.3%), followed by Manu (3/95, 3.2%) and S (1/95, 1%) lineages. Four of the six Beijing family isolates (66.7%) were MDR. A combination of IS6110-RFLP and spoligotyping reduced the clustering rate from 79% to 11.5% among the drug resistant isolates.
Conclusions
The results obtained showed that ill-defined T, Haarlem, the Turkey family (previously designated as LAM7-TUR family with high phylogeographical specifity for Turkey), Beijing and LAM were predominant lineages observed in almost 80% of the drug-Resistant M. tuberculosis complex clinical isolates in Ankara, Turkey.
doi:10.1371/journal.pone.0030331
PMCID: PMC3261197  PMID: 22279583
19.  Finer Snapshot of Circulating Mycobacterium tuberculosis Genotypes in Guadeloupe, Martinique, and French Guiana▿† 
Journal of Clinical Microbiology  2011;49(7):2685-2687.
This study shows the benefit of spoligotyping coupled to mycobacterial interspersed repetitive-unit (MIRU) typing to pinpoint circulating Mycobacterium tuberculosis genotypes in Guadeloupe, Martinique, and French Guiana. We hereby propose reduced 4-locus and 6-locus subsets for LAM and Haarlem lineage strains that predominate in South America and the Caribbean, retaining 99.35% and 99.64% of the total discriminatory power of the 12-locus scheme, respectively.
doi:10.1128/JCM.00708-11
PMCID: PMC3147841  PMID: 21562111
21.  Impact of Immigration on the Molecular Epidemiology of Tuberculosis in Rhode Island▿ 
Journal of Clinical Microbiology  2011;49(3):834-844.
While foreign-born persons constitute only 11% of the population in the state of Rhode Island, they account for more than 65% of incident tuberculosis (TB) annually. We investigated the molecular-epidemiological differences between foreign-born and U.S.-born TB patients to estimate the degree of recent transmission and identify predictors of clustering. A total of 288 isolates collected from culture-confirmed TB cases in Rhode Island between 1995 and 2004 were fingerprinted by spoligotyping and 12-locus mycobacterial interspersed repetitive units. Of the 288 fingerprinted isolates, 109 (37.8%) belonged to 36 genetic clusters. Our findings demonstrate that U.S.-born patients, Hispanics, Asian/Pacific islanders, and uninsured patients were significantly more likely to be clustered. Recent transmission among the foreign-born population was restricted and occurred mostly locally, within populations originating from the same region. Nevertheless, TB transmission between the foreign-born and U.S.-born population should not be neglected, since 80% of the mixed clusters of foreign- and U.S.-born persons arose from a foreign-born source case. We conclude that timely access to routine screening and treatment for latent TB infection for immigrants is vital for disease elimination in Rhode Island.
doi:10.1128/JCM.01952-10
PMCID: PMC3067685  PMID: 21159930
22.  Mycobacterium tuberculosis Complex Genotype Diversity and Drug Resistance Profiles in a Pediatric Population in Mexico 
The aim of this study was to determine the frequency of drug resistance and the clonality of genotype patterns in M. tuberculosis clinical isolates from pediatric patients in Mexico (n = 90 patients from 19 states; time period—January 2002 to December 2003). Pulmonary disease was the most frequent clinical manifestation (71%). Children with systemic tuberculosis (TB) were significantly younger compared to patients with localized TB infections (mean 7.7 ± 6.2 years versus 15 ± 3.4 years P = 0.001). Resistance to any anti-TB drug was detected in 24/90 (26.7%) of the isolates; 21/90 (23.3%) and 10/90 (11.1%) were resistant to Isoniazid and Rifampicin, respectively, and 10/90 (11.1%) strains were multidrug-resistant (MDR). Spoligotyping produced a total of 55 different patterns; 12/55 corresponded to clustered isolates (n = 47, clustering rate of 52.2%), and 43/55 to unclustered isolates (19 patterns were designated as orphan by the SITVIT2 database). Database comparison led to designation of 36 shared types (SITs); 32 SITs (n = 65 isolates) matched a preexisting shared type in SITVIT2, whereas 4 SITs (n = 6 isolates) were newly created. Lineage classification based on principal genetic groups (PGG) revealed that 10% of the strains belonged to PGG1 (Bovis and Manu lineages). Among PGG2/3 group, the most predominant clade was the Latin-American and Mediterranean (LAM) in 27.8% of isolates, followed by Haarlem and T lineages. The number of single drug-resistant (DR) and multidrug-resistant (MDR-TB) isolates in this study was similar to previously reported in studies from adult population with risk factors. No association between the spoligotype, age, region, or resistance pattern was observed. However, contrary to a study on M. tuberculosis spoligotyping in Acapulco city that characterized a single cluster of SIT19 corresponding to the EAI2-Manila lineage in 70 (26%) of patients, not a single SIT19 isolate was found in our pediatric patient population. Neither did we find any shared type belonging to the EAI family which represents ancestral PGG1 strains within the M. tuberculosis complex. We conclude that the population structure of pediatric TB in our setting is different from the one prevailing in adult TB patient population of Guerrero.
doi:10.1155/2011/239042
PMCID: PMC3335619  PMID: 22567263
23.  Spoligotype Profile of Mycobacterium tuberculosis Complex Strains from HIV-Positive and -Negative Patients in Nigeria: a Comparative Analysis ▿  
Journal of Clinical Microbiology  2010;49(1):220-226.
We ran a comparative analysis of all patients for whom a positive culture of Mycobacterium tuberculosis complex was available between April 2004 and October 2005 and whose HIV serology results were known, with spoligotyping results (n = 163) split into 49 HIV-positive patients and 114 HIV-negative patients. Spoligotype international type 373 (SIT373) (T1 lineage), which was highly prevalent among the HIV+ patients, was totally absent from the HIV− population, suggesting that we had a specific clone affecting nearly 1/3 of all HIV-tuberculosis (TB)-coinfected patients. Among the LAM10-CAM sublineage strains, we had only a single strain of SIT403 among HIV− patients (0.88%), as opposed to 12.25% of the HIV+ population (χ2 = 10.77; P < 0.01), indicating a strong association between the strain and the HIV+ population. The LAM10-CAM lineage spoligotype SIT61 was prevalent among the 2 subsets (37.72% in HIV− versus 12.24% in HIV+ populations), though, with a significant difference between the 2 groups (χ2 = 10.53; P < 0.01). However, there was no significant difference for SIT53 (T1 lineage) in the 2 subsets: 6.14 versus 8.2% (χ2 = 0.22; P > 0.05). A total of 7/49, or 14.3%, other SITs among HIV+ patients were not found among the HIV− patients. When added to the most prevalent SIT among HIV+ patients (SIT373; n = 16), 23/49, or 47%, isolates among HIV-TB-coinfected patients were unique. We conclude that further studies should be carried out to investigate the evolution of these genotypes and others in the emergence of multidrug resistance and control of tuberculosis in Nigeria.
doi:10.1128/JCM.01241-10
PMCID: PMC3020480  PMID: 21048016
24.  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.
doi:10.1128/JCM.00572-10
PMCID: PMC3020827  PMID: 20810763
25.  The use of microbead-based spoligotyping for Mycobacterium tuberculosis complex to evaluate the quality of the conventional method: Providing guidelines for Quality Assurance when working on membranes 
BMC Infectious Diseases  2011;11:110.
Background
The classical spoligotyping technique, relying on membrane reverse line-blot hybridization of the spacers of the Mycobacterium tuberculosis CRISPR locus, is used world-wide (598 references in Pubmed on April 8th, 2011). However, until now no inter-laboratory quality control study had been undertaken to validate this technique. We analyzed the quality of membrane-based spoligotyping by comparing it to the recently introduced and highly robust microbead-based spoligotyping. Nine hundred and twenty-seven isolates were analyzed totaling 39,861 data points. Samples were received from 11 international laboratories with a worldwide distribution.
Methods
The high-throughput microbead-based Spoligotyping was performed on CTAB and thermolyzate DNA extracted from isolated Mycobacterium tuberculosis complex (MTC) strains coming from the genotyping participating centers. Information regarding how the classical Spoligotyping method was performed by center was available. Genotype discriminatory analyses were carried out by comparing the spoligotypes obtained by both methods. The non parametric U-Mann Whitney homogeneity test and the Spearman rank correlation test were performed to validate the observed results.
Results
Seven out of the 11 laboratories (63 %), perfectly typed more than 90% of isolates, 3 scored between 80-90% and a single center was under 80% reaching 51% concordance only. However, this was mainly due to discordance in a single spacer, likely having a non-functional probe on the membrane used. The centers using thermolyzate DNA performed as well as centers using the more extended CTAB extraction procedure. Few centers shared the same problematic spacers and these problematic spacers were scattered over the whole CRISPR locus (Mostly spacers 15, 14, 18, 37, 39, 40).
Conclusions
We confirm that classical spoligotyping is a robust method with generally a high reliability in most centers. The applied DNA extraction procedure (CTAB or thermolyzate) did not affect the results in this study. However performance was center-dependent, suggesting that training is a key component in quality assurance of spoligotyping. Overall, no particular spacer yielded a higher degree of deviating results, suggesting that errors occur randomly either in the process of re-using membranes, or during the reading of the results and transferring of data from the film to a digital file. Last, the performance of the microbead-based method was excellent as previously shown by Cowan et al. (J. Clin. Microbiol. 2004) and Zhang et al. (J. Med. Microbiol. 2009) and demonstrated the proper detection of spacer 15 that is known to occasionally give weak signals in the classical spoligotyping.
doi:10.1186/1471-2334-11-110
PMCID: PMC3107175  PMID: 21527037

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