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1.  Molecular Epidemiology of HIV-Associated Tuberculosis in Dar es Salaam, Tanzania: Strain Predominance, Clustering, and Polyclonal Disease 
Journal of Clinical Microbiology  2012;50(8):2645-2650.
Molecular typing of Mycobacterium tuberculosis can be used to elucidate the epidemiology of tuberculosis, including the rates of clustering, the frequency of polyclonal disease, and the distribution of genotypic families. We performed IS6110 typing and spoligotyping on M. tuberculosis strains isolated from HIV-infected subjects at baseline or during follow-up in the DarDar Trial in Tanzania and on selected community isolates. Clustering occurred in 203 (74%) of 275 subjects: 124 (80%) of 155 HIV-infected subjects with baseline isolates, 56 (69%) of 81 HIV-infected subjects with endpoint isolates, and 23 (59%) of 39 community controls. Overall, 113 (41%) subjects had an isolate representing the East Indian “GD” family. The rate of clustering was similar among vaccine and placebo recipients and among subjects with or without cellular immune responses to mycobacterial antigens. Polyclonal disease was detected in 6 (43%) of 14 patients with multiple specimens typed. Most cases of HIV-associated tuberculosis among subjects from this study in Dar es Salaam resulted from recently acquired infection. Polyclonal infection was detected and isolates representing the East Indian GD strain family were the most common.
doi:10.1128/JCM.00624-12
PMCID: PMC3421504  PMID: 22649022
2.  Spoligologos: A Bioinformatic Approach to Displaying and Analyzing Mycobacterium tuberculosis Data 
Emerging Infectious Diseases  2002;8(11):1306-1309.
Spacer oligonucleotide (spoligotyping) analysis is a rapid polymerase chain reaction–based method of DNA fingerprinting the Mycobacterium tuberculosis complex. We examined spoligotype data using a bioinformatic tool (sequence logo analysis) to elucidate undisclosed phylogenetic relationships and gain insights into the global dissemination of strains of tuberculosis. Logo analysis of spoligotyping data provides a simple way to describe a fingerprint signature and may be useful in categorizing unique spoligotypes patterns as they are discovered. Large databases of DNA fingerprint information, such as those from the U.S. National Tuberculosis Genotyping and Surveillance Network and the European Concerted Action on Tuberculosis, contain information on thousands of strains from diverse regions. The description of related spoligotypes has depended on exhaustive listings of the individual spoligotyping patterns. Logo analysis may become another useful graphic method of visualizing and presenting spoligotyping clusters from these databases.
doi:10.3201/eid0811.020174
PMCID: PMC2738554  PMID: 12453361
tuberculosis; DNA fingerprinting; bioinformatics; spoligotyping
3.  Universal Genotyping in Tuberculosis Control Program, New York City, 2001–2003 
Emerging Infectious Diseases  2006;12(5):719-724.
Real-time universal genotyping decreased unnecessary treatment.
In 2001, New York City implemented genotyping to its tuberculosis (TB) control activities by using IS6110 restriction fragment length polymorphism (RFLP) and spoligotyping to type isolates from culture-positive TB patients. Results are used to identify previously unknown links among genotypically clustered patients, unidentified sites of transmission, and potential false-positive cultures. From 2001 to 2003, spoligotype and IS6110-based RFLP results were obtained for 90.7% of eligible and 93.7% of submitted isolates. Fifty-nine (2.4%) of 2,437 patient isolates had false-positive culture results, and 205 genotype clusters were identified, with 2–81 cases per cluster. Cluster investigations yielded 57 additional links and 17 additional sites of transmission. Four additional TB cases were identified as a result of case finding initiated through cluster investigations. Length of unnecessary treatment decreased among patients with false-positive cultures.
doi:10.3201/eid1205.050446
PMCID: PMC3374450  PMID: 16704826
Genotyping; tuberculosis; epidemiology; transmission; perspective
4.  Mycobacterium tuberculosis complex genetic diversity: mining the fourth international spoligotyping database (SpolDB4) for classification, population genetics and epidemiology 
BMC Microbiology  2006;6:23.
Background
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.
Results
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.
Conclusion
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.
doi:10.1186/1471-2180-6-23
PMCID: PMC1468417  PMID: 16519816
5.  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.
doi:10.1128/JCM.41.5.1963-1970.2003
PMCID: PMC154710  PMID: 12734235
6.  Impact of Genotyping of Mycobacterium tuberculosis on Public Health Practice in Massachusetts 
Emerging Infectious Diseases  2002;8(11):1285-1289.
Massachusetts was one of seven sentinel surveillance sites in the National Tuberculosis Genotyping and Surveillance Network. From 1996 through 2000, isolates from new patients with tuberculosis (TB) underwent genotyping. We describe the impact that genotyping had on public health practice in Massachusetts and some limitations of the technique. Through genotyping, we explored the dynamics of TB outbreaks, investigated laboratory cross-contamination, and identified Mycobacterium tuberculosis strains, transmission sites, and accurate epidemiologic links. Genotyping should be used with epidemiologic follow-up to identify how resources can best be allocated to investigate genotypic findings.
doi:10.3201/eid0811.020316
PMCID: PMC2738536  PMID: 12453357
Public health; tuberculosis; epidemiology; polymorphism; restriction fragment length; DNA fingerprinting; epidemiology; molecular
7.  Global Distribution of Mycobacterium tuberculosis Spoligotypes 
Emerging Infectious Diseases  2002;8(11):1347-1349.
We present a short summary of recent observations on the global distribution of the major clades of the Mycobacterium tuberculosis complex, the causative agent of tuberculosis. This global distribution was defined by data-mining of an international spoligotyping database, SpolDB3. This database contains 11,708 patterns from as many clinical isolates originating from more than 90 countries. The 11,708 spoligotypes were clustered into 813 shared types. A total of 1,300 orphan patterns (clinical isolates showing a unique spoligotype) were also detected.
doi:10.3201/eid0811.020125
PMCID: PMC2738532  PMID: 12453368
Mycobacterium tuberculosis; spoligotyping

Results 1-8 (8)