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1.  Tuberculosis-Spoligo-Rifampin-Isoniazid Typing: an All-in-One Assay Technique for Surveillance and Control of Multidrug-Resistant Tuberculosis on Luminex Devices 
Journal of Clinical Microbiology  2013;51(11):3527-3534.
As a follow-up of the “spoligoriftyping” development, we present here an extension of this technique which includes the detection of isoniazid resistance-associated mutations in a new 59-plex assay, i.e., tuberculosis-spoligo-rifampin-isoniazid typing (TB-SPRINT), running on microbead-based multiplexed systems. This assay improves the synergy between clinical microbiology and epidemiology by providing (i) mutation-based prediction of drug resistance profiles for patient treatment and (ii) genotyping data for tuberculosis (TB) surveillance. This third-generation microbead-based high-throughput assay for TB runs on the Luminex 200 system and on the recently launched MagPix system (Luminex, Austin, TX). Spoligotyping patterns obtained by the TB-SPRINT method were 100% (n = 85 isolates; 3,655/3,655 spoligotype data points) concordant with those obtained by microbead-based and membrane-based spoligotyping. Genetic drug susceptibility typing provided by the TB-SPRINT method was 100% concordant with resistance locus sequencing (n = 162 for rpoB gene sequencing and n = 76 for katG and inhA sequencing). Considering phenotypic drug susceptibility testing (DST) as the reference method, the sensitivity and specificity of TB-SPRINT regarding Mycobacterium tuberculosis complex (n = 162 isolates) rifampin resistance were both 100%, and those for isoniazid resistance were 90.4% (95% confidence interval, 85 to 95%) and 100%, respectively. Used routinely in national TB reference and specialized laboratories, the TB-SPRINT assay should simultaneously improve personalized medicine and epidemiological surveillance of multidrug-resistant (MDR) TB. This assay is expected to play an emerging role in public health in countries with heavy burdens of MDR TB and/or HIV/TB coinfection. Application of this assay directly to biological samples, as well as development for extensively drug-resistant (XDR) TB detection by inclusion of second-line antituberculosis drug-associated mutations, is under development. With bioinformatical methods and data mining to reduce the number of targets to the most informative ones, locally adapted formats of this technique can easily be developed everywhere.
doi:10.1128/JCM.01523-13
PMCID: PMC3889729  PMID: 23966495
2.  “Spoligoriftyping,” a Dual-Priming-Oligonucleotide-Based Direct-Hybridization Assay for Tuberculosis Control with a Multianalyte Microbead-Based Hybridization System 
Journal of Clinical Microbiology  2012;50(10):3172-3179.
We developed “spoligoriftyping,” a 53-plex assay based on two preexisting methods, the spoligotyping and “rifoligotyping” assays, by combining them into a single assay. Spoligoriftyping allows simultaneous spoligotyping (i.e., clustered regularly interspaced short palindromic repeat [CRISPR]-based genotyping) and characterization of the main rifampin drug resistance mutations on the rpoB hot spot region in a few hours. This test partly uses the dual-priming-oligonucleotide (DPO) principle, which allows simultaneous efficient amplifications of rpoB and the CRISPR locus in the same sample. We tested this method on a set of 114 previously phenotypically and genotypically characterized multidrug-resistant (MDR) Mycobacterium tuberculosis or drug-susceptible M. tuberculosis DNA extracted from clinical isolates obtained from patients from Bulgaria, Nigeria, and Germany. We showed that our method is 100% concordant with rpoB sequencing results and 99.95% (3,911/3,913 spoligotype data points) correlated with classical spoligotyping results. The sensitivity and specificity of our assay were 99 and 100%, respectively, compared to those of phenotypic drug susceptibility testing. Such assays pave the way to the implementation of locally and specifically adapted methods of performing in a single tube both drug resistance mutation detection and genotyping in a few hours.
doi:10.1128/JCM.00976-12
PMCID: PMC3457427  PMID: 22814456
4.  Combined Species Identification, Genotyping, and Drug Resistance Detection of Mycobacterium tuberculosis Cultures by MLPA on a Bead-Based Array 
PLoS ONE  2012;7(8):e43240.
The population structure of Mycobacterium tuberculosis is typically clonal therefore genotypic lineages can be unequivocally identified by characteristic markers such as mutations or genomic deletions. In addition, drug resistance is mainly mediated by mutations. These issues make multiplexed detection of selected mutations potentially a very powerful tool to characterise Mycobacterium tuberculosis. We used Multiplex Ligation-dependent Probe Amplification (MLPA) to screen for dispersed mutations, which can be successfully applied to Mycobacterium tuberculosis as was previously shown. Here we selected 47 discriminative and informative markers and designed MLPA probes accordingly to allow analysis with a liquid bead array and robust reader (Luminex MAGPIX technology). To validate the bead-based MLPA, we screened a panel of 88 selected strains, previously characterised by other methods with the developed multiplex assay using automated positive and negative calling. In total 3059 characteristics were screened and 3034 (99.2%) were consistent with previous molecular characterizations, of which 2056 (67.2%) were directly supported by other molecular methods, and 978 (32.0%) were consistent with but not directly supported by previous molecular characterizations. Results directly conflicting or inconsistent with previous methods, were obtained for 25 (0.8%) of the characteristics tested. Here we report the validation of the bead-based MLPA and demonstrate its potential to simultaneously identify a range of drug resistance markers, discriminate the species within the Mycobacterium tuberculosis complex, determine the genetic lineage and detect and identify the clinically most relevant non-tuberculous mycobacterial species. The detection of multiple genetic markers in clinically derived Mycobacterium tuberculosis strains with a multiplex assay could reduce the number of TB-dedicated screening methods needed for full characterization. Additionally, as a proportion of the markers screened are specific to certain Mycobacterium tuberculosis lineages each profile can be checked for internal consistency. Strain characterization can allow selection of appropriate treatment and thereby improve treatment outcome and patient management.
doi:10.1371/journal.pone.0043240
PMCID: PMC3423362  PMID: 22916230
5.  A Molecular Epidemiological and Genetic Diversity Study of Tuberculosis in Ibadan, Nnewi and Abuja, Nigeria 
PLoS ONE  2012;7(6):e38409.
Background
Nigeria has the tenth highest burden of tuberculosis (TB) among the 22 TB high-burden countries in the world. This study describes the biodiversity and epidemiology of drug-susceptible and drug-resistant TB in Ibadan, Nnewi and Abuja, using 409 DNAs extracted from culture positive TB isolates.
Methodology/Principal Findings
DNAs extracted from clinical isolates of Mycobacterium tuberculosis complex were studied by spoligotyping and 24 VNTR typing. The Cameroon clade (CAM) was predominant followed by the M. africanum (West African 1) and T (mainly T2) clades. By using a smooth definition of clusters, 32 likely epi-linked clusters related to the Cameroon genotype family and 15 likely epi-linked clusters related to other “modern” genotypes were detected. Eight clusters concerned M. africanum West African 1. The recent transmission rate of TB was 38%. This large study shows that the recent transmission of TB in Nigeria is high, without major regional differences, with MDR-TB clusters. Improvement in the TB control programme is imperative to address the TB control problem in Nigeria.
doi:10.1371/journal.pone.0038409
PMCID: PMC3377642  PMID: 22723859
6.  CRISPR Typing and Subtyping for Improved Laboratory Surveillance of Salmonella Infections 
PLoS ONE  2012;7(5):e36995.
Laboratory surveillance systems for salmonellosis should ideally be based on the rapid serotyping and subtyping of isolates. However, current typing methods are limited in both speed and precision. Using 783 strains and isolates belonging to 130 serotypes, we show here that a new family of DNA repeats named CRISPR (clustered regularly interspaced short palindromic repeats) is highly polymorphic in Salmonella. We found that CRISPR polymorphism was strongly correlated with both serotype and multilocus sequence type. Furthermore, spacer microevolution discriminated between subtypes within prevalent serotypes, making it possible to carry out typing and subtyping in a single step. We developed a high-throughput subtyping assay for the most prevalent serotype, Typhimurium. An open web-accessible database was set up, providing a serotype/spacer dictionary and an international tool for strain tracking based on this innovative, powerful typing and subtyping tool.
doi:10.1371/journal.pone.0036995
PMCID: PMC3356390  PMID: 22623967
7.  Spoligotyping of Mycobacterium africanum, Burkina Faso 
Emerging Infectious Diseases  2012;18(1):117-119.
Using Ziehl-Neelsen–positive slides collected from tuberculosis diagnostic centers in Burkina Faso, we showed that 20% of 80 spoligotyping-positive DNA samples had a characteristic Mycobacterium africanum–specific genomic signature. This result suggests that M. africanum is still present in Burkina Faso at almost the same prevalence as 15–20 years ago.
doi:10.3201/eid1801.110275
PMCID: PMC3310091  PMID: 22257494
Mycobacterium africanum; Burkina Faso; spoligotyping; bacteria; tuberculosis and other mycobacteria
8.  Using affinity propagation for identifying subspecies among clonal organisms: lessons from M. tuberculosis 
BMC Bioinformatics  2011;12:224.
Background
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.
Results
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.
Conclusion
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.
doi:10.1186/1471-2105-12-224
PMCID: PMC3126747  PMID: 21635750
asexual organisms; species delineation; epidemiology; DR locus; Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)
9.  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
10.  A first assessment of the genetic diversity of Mycobacterium tuberculosis complex in Cambodia 
Background
Cambodia is among the 22 high-burden TB countries, and has one of the highest rates of TB in South-East Asia. This study aimed to describe the genetic diversity among clinical Mycobacterium tuberculosis complex (MTC) isolates collected in Cambodia and to relate these findings to genetic diversity data from neighboring countries.
Methods
We characterized by 24 VNTR loci genotyping and spoligotyping 105 Mycobacterium tuberculosis clinical isolates collected between 2007 and 2008 in the region of Phnom-Penh, Cambodia, enriched in multidrug-resistant (MDR) isolates (n = 33).
Results
Classical spoligotyping confirmed that the East-African Indian (EAI) lineage is highly prevalent in this area (60%-68% respectively in whole sample and among non-MDR isolates). Beijing lineage is also largely represented (30% in whole sample, 21% among non-MDR isolates, OR = 4.51, CI95% [1.77, 11.51]) whereas CAS lineage was absent. The 24 loci MIRU-VNTR typing scheme distinguished 90 patterns with only 13 multi-isolates clusters covering 28 isolates. The clustering of EAI strains could be achieved with only 8 VNTR combined with spoligotyping, which could serve as a performing, easy and cheap genotyping standard for this family. Extended spoligotyping suggested relatedness of some unclassified "T1 ancestors" or "Manu" isolates with modern strains and provided finer resolution.
Conclusions
The genetic diversity of MTC in Cambodia is driven by the EAI and the Beijing families. We validate the usefulness of the extended spoligotyping format in combination with 8 VNTR for EAI isolates in this region.
doi:10.1186/1471-2334-11-42
PMCID: PMC3062598  PMID: 21299851
11.  First Insight into Genetic Diversity of the Mycobacterium tuberculosis Complex in Albania Obtained by Multilocus Variable-Number Tandem-Repeat Analysis and Spoligotyping Reveals the Presence of Beijing Multidrug-Resistant Isolates ▿  
Journal of Clinical Microbiology  2009;47(5):1581-1584.
We characterized a set of 100 Mycobacterium tuberculosis complex clinical isolates from tuberculosis (TB) patients in Albania, typing them with a 24-locus variable-number tandem-repeat-spoligotyping scheme. Depending on the cluster definition, 43 to 49 patients were distributed into 15 to 16 clusters which were likely to be epidemiologically linked, indicative of a recent transmission rate of 28 to 34%. This result suggests that TB is under control in Albania. However, two multidrug-resistant (MDR) Beijing genotypes harboring the same S531A mutation on the rpoB gene were also found, suggesting a potential recent transmission of MDR TB. Three brand new genotypes, Albania-1 to Albania-3, are also described.
doi:10.1128/JCM.02284-08
PMCID: PMC2681852  PMID: 19279172
12.  Application of Sensitive and Specific Molecular Methods To Uncover Global Dissemination of the Major RDRio Sublineage of the Latin American-Mediterranean Mycobacterium tuberculosis Spoligotype Family▿ ‡ 
Journal of Clinical Microbiology  2008;46(4):1259-1267.
The Latin American-Mediterranean (LAM) family of Mycobacterium tuberculosis is believed to be the cause of ∼15% of tuberculosis cases worldwide. Previously, we defined a prevalent sublineage of the LAM family in Brazil by a single characteristic genomic deletion designated RDRio. Using the Brazilian strains, we pinpoint an Ag85C103 single nucleotide polymorphism (SNP) (screened by restriction fragment length polymorphism [RFLP] analysis) that correctly identified all LAM family strains. Importantly, all RDRio strains concomitantly possessed the RD174 deletion. These genetic signatures, along with a newly developed multiplex PCR for rapid differentiation between “wild-type” and RDRio strains, were then used to analyze an international collection of M. tuberculosis strains. RDRio M. tuberculosis was identified from four continents involving 11 countries. Phylogenetic analysis of the IS6110-RFLP patterns from representative RDRio and LAM strains from Brazil, along with all representative clusters from a South African database, confirmed their genetic relatedness and transcontinental transmission. The Ag85C103 SNP RFLP, as compared to results obtained using a PCR method targeting a LAM-restricted IS6110 element, correctly identified 99.8% of LAM spoligotype strains. Together, these tests were more accurate than spoligotyping at categorizing strains with indefinable spoligotypes and segregated true LAM strains from those with convergent spoligotypes. The fact that RDRio strains were identified worldwide highlights the importance of this LAM family sublineage and suggests that this strain is a global threat that should be specifically targeted by public health resources. Our provision of simple and robust molecular methods will assist the evaluation of the LAM family and the RDRio sublineage.
doi:10.1128/JCM.02231-07
PMCID: PMC2292928  PMID: 18234868
13.  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 
Background
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.
Methods
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.
Results
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.
Conclusion
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.
doi:10.1186/1471-2334-8-46
PMCID: PMC2375894  PMID: 18410681
14.  Assessment of Population Structure and Major Circulating Phylogeographical Clades of Mycobacterium tuberculosis Complex in Bangladesh Suggests a High Prevalence of a Specific Subclade of Ancient M. tuberculosis Genotypes▿  
Journal of Clinical Microbiology  2007;45(11):3791-3794.
Spoligotyping was performed to study the population structure of Mycobacterium tuberculosis complex strains (n = 224) from Bangladesh. Strains were split into principal genetic group 1 (PGG 1 [75.0%]) and PGG 2 and 3 (25%). Forty-nine strains with a new spoligotype signature and considered as south or southeast Asian-linked emerging clones were designated as “Matlab type.”
doi:10.1128/JCM.01247-07
PMCID: PMC2168514  PMID: 17804653
15.  Assessment of Mycobacterial Interspersed Repetitive Unit-QUB Markers To Further Discriminate the Beijing Genotype in a Population-Based Study of the Genetic Diversity of Mycobacterium tuberculosis Clinical Isolates from Okinawa, Ryukyu Islands, Japan▿ †  
Journal of Clinical Microbiology  2007;45(11):3606-3615.
The present investigation focused on genetic diversity and drug resistance of 101 Mycobacterium tuberculosis strains isolated between July 2003 and February 2005 in the Okinawa prefecture, Ryukyu Islands, Japan. A high rate of clustering (87%, eight clusters, 2 to 69 strains/cluster) was observed upon spoligotyping; most of it was due to the lower discriminatory power of this method for the Beijing lineage (n = 72; 71.3% of the isolates). The remaining diversity was limited to seven clusters (two to five isolates/cluster), with the following distribution of major lineages: ill-defined T (n = 13; 12.8%), ancestral East African-Indian (n = 6; 5.9%), Haarlem (n = 4; 4%), Latin American-Mediterranean (n = 2; 2%), X1 (n = 1; 1%), and a total absence of the central Asian clade. Three remaining strains could not be classified on the basis of their spoligotype pattern and were labeled “unknown.” Subtyping with mycobacterial interspersed repetitive units (MIRUs) in association with additional QUB minisatellites was performed to discriminate among the Beijing strains. Based on an “in-house” spoligotyping/MIRU database (n = 694 Beijing strains), eight highly discriminative MIRU loci for Beijing strains were selected (loci numbered 10, 16, 23, 26, 27, 31, 39, and 40). The highest discriminatory power (h) observed in our sample (n = 72; M-26, 0.385; M-10, 0.38; M-31, 0.255; M-16, 0.238) was too low, and 73.6% of the Beijing strains from Okinawa remained clustered. Typing of Beijing strains with additional QUB loci (with the exception of “one-copy” QUB-1451) resulted in higher discriminatory powers: QUB-11b, 0.68; QUB-11a, 0.656; QUB-26, 0.644; QUB-18, 0.553; QUB-4156, 0.5; and QUB-1895, 0.453. A definitive algorithm on the use of QUB markers to subtype Beijing isolates in expanded studies would shed light on their hypervariability, which may sometimes blur recognition between epidemiologically linked Beijing isolates. The total absence of multiple drug resistance among Beijing isolates from Okinawa, as well as the relatively older ages of the patients (majority above 60 years), shows that tuberculosis (TB) is a declining disease in Okinawa, and an adequate TB control program has successfully avoided both the emergence and the spread of multidrug-resistant TB in this insular setting.
doi:10.1128/JCM.00348-07
PMCID: PMC2168487  PMID: 17898160
16.  Evolution and Diversity of Clonal Bacteria: The Paradigm of Mycobacterium tuberculosis 
PLoS ONE  2008;3(2):e1538.
Background
Mycobacterium tuberculosis complex species display relatively static genomes and 99.9% nucleotide sequence identity. Studying the evolutionary history of such monomorphic bacteria is a difficult and challenging task.
Principal Findings
We found that single-nucleotide polymorphism (SNP) analysis of DNA repair, recombination and replication (3R) genes in a comprehensive selection of M. tuberculosis complex strains from across the world, yielded surprisingly high levels of polymorphisms as compared to house-keeping genes, making it possible to distinguish between 80% of clinical isolates analyzed in this study. Bioinformatics analysis suggests that a large number of these polymorphisms are potentially deleterious. Site frequency spectrum comparison of synonymous and non-synonymous variants and Ka/Ks ratio analysis suggest a general negative/purifying selection acting on these sets of genes that may lead to suboptimal 3R system activity. In turn, the relaxed fidelity of 3R genes may allow the occurrence of adaptive variants, some of which will survive. Furthermore, 3R-based phylogenetic trees are a new tool for distinguishing between M. tuberculosis complex strains.
Conclusions/Significance
This situation, and the consequent lack of fidelity in genome maintenance, may serve as a starting point for the evolution of antibiotic resistance, fitness for survival and pathogenicity, possibly conferring a selective advantage in certain stressful situations. These findings suggest that 3R genes may play an important role in the evolution of highly clonal bacteria, such as M. tuberculosis. They also facilitate further epidemiological studies of these bacteria, through the development of high-resolution tools. With many more microbial genomes being sequenced, our results open the door to 3R gene-based studies of adaptation and evolution of other, highly clonal bacteria.
doi:10.1371/journal.pone.0001538
PMCID: PMC2211405  PMID: 18253486
17.  First Insight into the Population Structure of Mycobacterium tuberculosis in Saudi Arabia▿ †  
Journal of Clinical Microbiology  2007;45(8):2467-2473.
This study constitutes a first attempt to describe the genetic population structure and drug resistance of the tubercle bacilli circulating in Saudi Arabia. A total of 1,505 clinical isolates of M. tuberculosis, isolated between 2002 and 2005 from seven regions of Saudi Arabia, were studied. The sample studied showed a male-to-female sex ratio of 1.27, with half of the cases among foreign-born individuals and 47% within the 21- to 40-year-old age group; a total resistance rate of 19.7%; and multiple drug resistance of 4.5%. Upon spoligotyping, a total of 387 individual patterns were obtained (clustering rate, 86.4%; 182 clusters containing between 2 and 130 isolates per cluster). A total of 94% of the strains matched the spoligotype patterns in an international database. Nearly 81% of the isolates in this study belonged to established phylogeographic clades: Central Asian (CAS), 22.5%; ill-defined T clade, 19.5%; East African-Indian (EAI), 13.5%; Haarlem, 7.5%; Latin American-Mediterranean, 7.2%; Beijing, 4.4%; Manu, 2.7%; X, 0.9%; and Bovis, 0.9%. Two clonal complexes with unique spoligotyping signatures (octal codes 703777707770371 and 467777377413771) specific to Saudi Arabia were identified. These belonged to the CAS and EAI clades, respectively, as confirmed upon secondary typing using mycobacterial interspersed repetitive units (MIRUs). The results obtained underline the predominance of historic clones of principal genetic group 1, which are responsible for roughly 45% of all tuberculosis cases in Saudi Arabia. The high rate of clustering observed might be an indication of rapid ongoing transmission within certain communities and/or subpopulations in Saudi Arabia; nonetheless, spoligotyping is known to overestimate clustering, and only a systematic second-line typing, such as MIRUs, coupled with a better tuberculosis registry and epidemiological investigations would allow us to know the exact rate of ongoing transmission and associated risk factors in Saudi Arabia.
doi:10.1128/JCM.02293-06
PMCID: PMC1951255  PMID: 17507515
18.  Three-Year Longitudinal Study of Genotypes of Mycobacterium tuberculosis Isolates in Tuscany, Italy▿  
Journal of Clinical Microbiology  2007;45(6):1851-1857.
The genetic diversity of 829 strains of Mycobacterium tuberculosis isolated during a 3-year period in Tuscany, Italy, a country with a low prevalence of tuberculosis, from 480 Italian-born and 349 foreign-born patients was determined by spoligotyping. The predominant spoligotype families were T (30.2% of isolates), Haarlem (19.9%), and the Latino-American and Mediterranean family (LAM) (11.2%); the remaining isolates were distributed among the Beijing (6.5%), S (4.2%), East Africa-India (EAI) (3.0%), Bovis (2.3%), Central Asia (CAS) (2.1%), Africanum (1.3%), and X (1.2%) families or were undefined (2.7%) or orphan (14.1%) isolates. Isolates of the families T, Haarlem, Bovis, and X were distributed among Italian- and foreign-born patients almost proportionally to the patients' numbers. Isolates of the LAM family were prevalent in foreign-born people (13.5%, versus 9.6% in Italian-born patients). Isolates of the S family were found almost exclusively in Italian-born patients, while strains of families EAI and CAS were isolated almost exclusively from foreign-born patients; Africanum isolates were all from African-born patients. The isolates of the Beijing family showed a trend to a steady increase during the survey. The prevalence of Beijing strains was 11.7% among foreign-born people and 2.7% among Italian-born patients. The Beijing strains were typed by the standardized IS6110 restriction fragment length polymorphism assay, which yielded a total of 38 distinct IS6110 patterns; 21 isolates (39.6%) occurred in six distinct clusters; of these, three contained two isolates and the other three contained four, five and six isolates, thus demonstrating that Beijing strains caused several tuberculosis outbreaks in the region. These findings indicate that transmission of Beijing strains between immigrants and the autochthonous population has occurred frequently and suggests an ongoing active transmission of the Beijing genotype in the region.
doi:10.1128/JCM.00170-07
PMCID: PMC1933082  PMID: 17460055
19.  M. tuberculosis genotypic diversity and drug susceptibility pattern in HIV- infected and non-HIV-infected patients in northern Tanzania 
BMC Microbiology  2007;7:51.
Background
Tuberculosis (TB) is a major health problem and HIV is the major cause of the increase in TB. Sub-Saharan Africa is endemic for both TB and HIV infection. Determination of the prevalence of M. tuberculosis strains and their drug susceptibility is important for TB control.
TB positive culture, BAL fluid or sputum samples from 130 patients were collected and genotyped. The spoligotypes were correlated with anti-tuberculous drug susceptibility in HIV-infected and non-HIV patients from Tanzania.
Results
One-third of patients were TB/HIV co-infected. Forty-seven spoligotypes were identified.
Fourteen isolates (10.8%) had new and unique spoligotypes while 116 isolates (89.2%) belonged to 33 known spoligotypes. The major spoligotypes contained nine clusters: CAS1-Kili 30.0%, LAM11- ZWE 14.6%, ND 9.2%, EAI 6.2%, Beijing 5.4%, T-undefined 4.6%, CAS1-Delhi 3.8%, T1 3.8% and LAM9 3.8%. Twelve (10.8%) of the 111 phenotypically tested strains were resistant to anti-TB drugs. Eight (7.2%) were monoresistant strains: 7 to isoniazid (INH) and one to streptomycin. Four strains (3.5%) were resistant to multiple drugs: one (0.9%) was resistant to INH and streptomycin and the other three (2.7%) were MDR strains: one was resistant to INH, rifampicin and ethambutol and two were resistant to all four anti-TB drugs. Mutation in the katG gene codon 315 and the rpoB hotspot region showed a low and high sensitivity, respectively, as predictor of phenotypic drug resistance.
Conclusion
CAS1-Kili and LAM11-ZWE were the most common families. Strains of the Beijing family and CAS1-Kili were not or least often associated with resistance, respectively. HIV status was not associated with spoligotypes, resistance or previous TB treatment.
doi:10.1186/1471-2180-7-51
PMCID: PMC1913919  PMID: 17540031
20.  Proposal for Standardization of Optimized Mycobacterial Interspersed Repetitive Unit-Variable-Number Tandem Repeat Typing of Mycobacterium tuberculosis▿ †  
Journal of Clinical Microbiology  2006;44(12):4498-4510.
Molecular typing based on 12 loci containing variable numbers of tandem repeats of mycobacterial interspersed repetitive units (MIRU-VNTRs) has been adopted in combination with spoligotyping as the basis for large-scale, high-throughput genotyping of Mycobacterium tuberculosis. However, even the combination of these two methods is still less discriminatory than IS6110 fingerprinting. Here, we define an optimized set of MIRU-VNTR loci with a significantly higher discriminatory power. The resolution and the stability/robustness of 29 loci were analyzed, using a total of 824 tubercle bacillus isolates, including representatives of the main lineages identified worldwide so far. Five loci were excluded for lack of robustness and/or stability in serial isolates or isolates from epidemiologically linked patients. The use of the 24 remaining loci increased the number of types by 40%—and by 23% in combination with spoligotyping—among isolates from cosmopolitan origins, compared to those obtained with the original set of 12 loci. Consequently, the clustering rate was decreased by fourfold—by threefold in combination with spoligotyping—under the same conditions. A discriminatory subset of 15 loci with the highest evolutionary rates was then defined that concentrated 96% of the total resolution obtained with the full 24-locus set. Its predictive value for evaluating M. tuberculosis transmission was found to be equal to that of IS6110 restriction fragment length polymorphism typing, as shown in a companion population-based study. This 15-locus system is therefore proposed as the new standard for routine epidemiological discrimination of M. tuberculosis isolates and the 24-locus system as a high-resolution tool for phylogenetic studies.
doi:10.1128/JCM.01392-06
PMCID: PMC1698431  PMID: 17005759
21.  Insights into the evolutionary history of tubercle bacilli as disclosed by genetic rearrangements within a PE_PGRS duplicated gene pair 
Background
The highly homologous PE_PGRS (Proline-glutamic acid_polymorphic GC-rich repetitive sequence) genes are members of the PE multigene family which is found only in mycobacteria. PE genes are particularly abundant within the genomes of pathogenic mycobacteria where they seem to have expanded as a result of gene duplication events. PE_PGRS genes are characterized by their high GC content and extensive repetitive sequences, making them prone to recombination events and genetic variability.
Results
Comparative sequence analysis of Mycobacterium tuberculosis genes PE_PGRS17 (Rv0978c) and PE_PGRS18 (Rv0980c) revealed a striking genetic variation associated with this typical tandem duplicate. In comparison to the M. tuberculosis reference strain H37Rv, the variation (named the 12/40 polymorphism) consists of an in-frame 12-bp insertion invariably accompanied by a set of 40 single nucleotide polymorphisms (SNPs) that occurs either in PE_PGRS17 or in both genes. Sequence analysis of the paralogous genes in a representative set of worldwide distributed tubercle bacilli isolates revealed data which supported previously proposed evolutionary scenarios for the M. tuberculosis complex (MTBC) and confirmed the very ancient origin of "M. canettii" and other smooth tubercle bacilli. Strikingly, the identified polymorphism appears to be coincident with the emergence of the post-bottleneck successful clone from which the MTBC expanded. Furthermore, the findings provide direct and clear evidence for the natural occurrence of gene conversion in mycobacteria, which appears to be restricted to modern M. tuberculosis strains.
Conclusion
This study provides a new perspective to explore the molecular events that accompanied the evolution, clonal expansion, and recent diversification of tubercle bacilli.
doi:10.1186/1471-2148-6-107
PMCID: PMC1762029  PMID: 17163995
22.  Molecular characterisation of Mycobacterium tuberculosis isolates in the First National Survey of Anti-tuberculosis Drug Resistance from Venezuela 
BMC Microbiology  2006;6:90.
Background
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).
Results
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.
Conclusion
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.
doi:10.1186/1471-2180-6-90
PMCID: PMC1621067  PMID: 17032442
23.  Long-Term Population-Based Genotyping Study of Mycobacterium tuberculosis Complex Isolates in the French Departments of the Americas†  
Journal of Clinical Microbiology  2006;44(1):183-191.
The three French overseas departments of the Americas are characterized both by insular (Guadeloupe and Martinique) and continental (French Guiana) settings with a tuberculosis case detection rate that varies from less than 10 per 100,000 per year in insular areas to an estimated incidence of more than 55 per 100,000 in French Guiana. Under a long-term genotyping program, more than three-fourths of all the Mycobacterium tuberculosis isolates (n = 744) received from the three settings were fingerprinted over a 10-year period (1994 to 2003) by spoligotyping and variable number of tandem DNA repeats (VNTRs) in order to understand the current trends in their detection rates, drug resistance, and groups and subpopulations at risk of contracting the disease and to pinpoint the circulating phylogeographical clades of the bacilli. The major difference in the study populations was the nationality of the patients, with a high percentage of immigrants from high-incidence neighboring countries in French Guiana and a low but increasing percentage in the French Caribbean. The rate of recent transmission was calculated to be 49.3% in French Guiana, compared to 27.2% and 16.9% in Guadeloupe and Martinique, respectively. At the phylogeographic level, 77.9% of the isolates studied belonged to four major clades (Haarlem, Latin-American and Mediterranean, T, and X) which are already reported from neighboring Caribbean islands in an international database and may underline potential interregional transmission events.
doi:10.1128/JCM.44.1.183-191.2006
PMCID: PMC1351934  PMID: 16390968
24.  Global Phylogeny of Mycobacterium tuberculosis Based on Single Nucleotide Polymorphism (SNP) Analysis: Insights into Tuberculosis Evolution, Phylogenetic Accuracy of Other DNA Fingerprinting Systems, and Recommendations for a Minimal Standard SNP Set†  
Journal of Bacteriology  2006;188(2):759-772.
We analyzed a global collection of Mycobacterium tuberculosis strains using 212 single nucleotide polymorphism (SNP) markers. SNP nucleotide diversity was high (average across all SNPs, 0.19), and 96% of the SNP locus pairs were in complete linkage disequilibrium. Cluster analyses identified six deeply branching, phylogenetically distinct SNP cluster groups (SCGs) and five subgroups. The SCGs were strongly associated with the geographical origin of the M. tuberculosis samples and the birthplace of the human hosts. The most ancestral cluster (SCG-1) predominated in patients from the Indian subcontinent, while SCG-1 and another ancestral cluster (SCG-2) predominated in patients from East Asia, suggesting that M. tuberculosis first arose in the Indian subcontinent and spread worldwide through East Asia. Restricted SCG diversity and the prevalence of less ancestral SCGs in indigenous populations in Uganda and Mexico suggested a more recent introduction of M. tuberculosis into these regions. The East African Indian and Beijing spoligotypes were concordant with SCG-1 and SCG-2, respectively; X and Central Asian spoligotypes were also associated with one SCG or subgroup combination. Other clades had less consistent associations with SCGs. Mycobacterial interspersed repetitive unit (MIRU) analysis provided less robust phylogenetic information, and only 6 of the 12 MIRU microsatellite loci were highly differentiated between SCGs as measured by GST. Finally, an algorithm was devised to identify two minimal sets of either 45 or 6 SNPs that could be used in future investigations to enable global collaborations for studies on evolution, strain differentiation, and biological differences of M. tuberculosis.
doi:10.1128/JB.188.2.759-772.2006
PMCID: PMC1347298  PMID: 16385065

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