Philip Supply

Publications of Philip Supply

• Online tools for polyphasic analysis of Mycobacterium tuberculosis complex genotyping data: Now and next.

  Authors: Thomas Weniger, Justina Krawczyk, Philip Supply, Dag Harmsen, Stefan Niemann

  Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases. 02/2012;

  Molecular diagnostics and genotyping of pathogens have become indispensable tools in clinical microbiology and disease surveillance. For isolates of the Mycobacterium tuberculosis complex (MTBC,Molecular diagnostics and genotyping of pathogens have become indispensable tools in clinical microbiology and disease surveillance. For isolates of the Mycobacterium tuberculosis complex (MTBC, causative agents of tuberculosis), multilocus variable number tandem repeat analysis (MLVA) targeting mycobacterial interspersed repetitive units (MIRU) has been internationally adopted as the new standard, portable, reproducible, and discriminatory typing method. Here, we review new sets of specialized web based bioinformatics tools that have become available for analyzing MLVA data especially in combination with other, complementary genotyping markers (polyphasic analysis). Currently, there are only two databases available that are not restricted to store one kind of genotyping data only, namely SITVIT/SpolDB4 and MIRU-VNTRplus. SITVIT/SpolDB4 (http://www.pasteur-guadeloupe.fr:8081/SITVITDemo) contains spoligotyping data from a large number of strains of diverse origin. However, besides options to query the data, the actual version of SITVIT/SpolDB4 offers no functionality for more complex analysis e.g. tree-based analysis. In comparison, the MIRU-VNTRplus web application (http://www.miru-vntrplus.org), represents a freely accessible service that enables users to analyze genotyping data of their strains alone or in comparison with a currently limited but well characterized reference database of strains representing the major MTBC lineages. Data (MLVA-, spoligotype-, large sequence polymorphism, and single nucleotide polymorphism) can be visualized and analyzed using just one genotyping method or a weighted combination of several markers. A variety of analysis tools are available such as creation of phylogenetic and minimum spanning trees, semi-automated phylogenetic lineage identification based on comparison with the reference database and mapping of geographic information. To facilitate scientific communication, a universal, expanding genotype nomenclature (MLVA MtbC15-9 type) service that can be queried via a web- or a SOAP-interface has been implemented. An extensive documentation guides users through all application functions. Perspectives for future development, including generalization to other bacterial species, are presented.

• First Worldwide Proficiency Study on Variable-Number Tandem-Repeat Typing of Mycobacterium tuberculosis Complex Strains.

  Authors: Jessica L de Beer, Kristin Kremer, Csaba Ködmön, Philip Supply, Dick van Soolingen

  Journal of clinical microbiology. 12/2011; 50(3):662-669.

  Although variable-number tandem-repeat (VNTR) typing has gained recognition as the new standard for the DNA fingerprinting of Mycobacterium tuberculosis complex (MTBC) isolates, external qualityAlthough variable-number tandem-repeat (VNTR) typing has gained recognition as the new standard for the DNA fingerprinting of Mycobacterium tuberculosis complex (MTBC) isolates, external quality control programs have not yet been developed. Therefore, we organized the first multicenter proficiency study on 24-locus VNTR typing. Sets of 30 DNAs of MTBC strains, including 10 duplicate DNA samples, were distributed among 37 participating laboratories in 30 different countries worldwide. Twenty-four laboratories used an in-house-adapted method with fragment sizing by gel electrophoresis or an automated DNA analyzer, nine laboratories used a commercially available kit, and four laboratories used other methods. The intra- and interlaboratory reproducibilities of VNTR typing varied from 0% to 100%, with averages of 72% and 60%, respectively. Twenty of the 37 laboratories failed to amplify particular VNTR loci; if these missing results were ignored, the number of laboratories with 100% interlaboratory reproducibility increased from 1 to 5. The average interlaboratory reproducibility of VNTR typing using a commercial kit was better (88%) than that of in-house-adapted methods using a DNA analyzer (70%) or gel electrophoresis (50%). Eleven laboratories using in-house-adapted manual typing or automated typing scored inter- and intralaboratory reproducibilities of 80% or higher, which suggests that these approaches can be used in a reliable way. In conclusion, this first multicenter study has documented the worldwide quality of VNTR typing of MTBC strains and highlights the importance of international quality control to improve genotyping in the future.

• One year nationwide evaluation of 24-locus MIRU-VNTR genotyping on Slovenian Mycobacterium tuberculosis isolates.

  Authors: Urska Bidovec-Stojkovic, Manca Zolnir-Dovc, Philip Supply

  Respiratory medicine. 10/2011; 105 Suppl 1:S67-73.

  Slovenia is one of the few countries where IS6110 RFLP is applied for genotyping M. tuberculosis at a nationwide level, which has been in effect since 2000. Based on S6110 RFLP clustering, typicalSlovenia is one of the few countries where IS6110 RFLP is applied for genotyping M. tuberculosis at a nationwide level, which has been in effect since 2000. Based on S6110 RFLP clustering, typical risk factors and routes of M. tuberculosis transmission were identified, such as alcohol abuse, homelessness, and bars. However, IS6110 RFLP typing suffers from important limitations including a long wait for results, which reduces the potential benefit of molecular-guided tuberculosis (TB) control. PCR-based 24-locus MIRU-VNTR typing combined with spoligotyping has recently emerged as a potential alternative for faster, large-scale genotyping of M. tuberculosis. We compared these genotyping methods for analyzing 196 Slovenian Mycobacterium tuberculosis isolates representing 97.5% of all culture-positive cases included in the Slovenian TB Registry in 2008. IS6110 RFLP and 24-locus MIRU-VNTR typing combined with spoligotyping identified 157 and 155 distinct profiles, 135 and 125 unique isolates, and 61 and 71 clustered isolates grouped into 22 and 29 clusters, respectively. The discriminatory indexes were very close, at 0.9963 and 0.9965, respectively. The majority of the molecular clusters defined by either of the two methods were identical, including in the few cases for which epidemiological links were available. The differences frequently consisted of single-band changes in IS6170-RFLP profiles subdividing a MIRU-VNTR/spoligotype-based cluster. Our one-year nationwide study showed that the results of 24-locus MIRU-VNTR typing combined with spoligotyping reached a high level of concordance with those obtained from IS6110 RFLP typing.

• On the mutation rates of spoligotypes and variable numbers of tandem repeat loci of Mycobacterium tuberculosis: continued-When tuning matters.

  Authors: Philip Supply, Stefan Niemann, Thierry Wirth

  Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases. 03/2011;

• The forest behind the tree: phylogenetic exploration of a dominant Mycobacterium tuberculosis strain lineage from a high tuberculosis burden country.

  Authors: Maranibia Cardoso Oelemann, Harrison M Gomes, Eve Willery, Lia Possuelo, Karla Valéria Batista Lima, Caroline Allix-Béguec, Camille Locht, Yves-Olivier L Goguet de la Salmonière, Maria Cristina Gutierrez, Philip Suffys, Philip Supply

  PloS one. 01/2011; 6(3):e18256.

  Genotyping of Mycobacterium tuberculosis isolates is a powerful tool for epidemiological control of tuberculosis (TB) and phylogenetic exploration of the pathogen. Standardized PCR-based typing,Genotyping of Mycobacterium tuberculosis isolates is a powerful tool for epidemiological control of tuberculosis (TB) and phylogenetic exploration of the pathogen. Standardized PCR-based typing, based on 15 to 24 mycobacterial interspersed repetitive unit-variable number of tandem repeat (MIRU-VNTR) loci combined with spoligotyping, has been shown to have adequate resolution power for tracing TB transmission and to be useful for predicting diverse strain lineages in European settings. Its informative value needs to be tested in high TB-burden countries, where the use of genotyping is often complicated by dominance of geographically specific, genetically homogeneous strain lineages. We tested this genotyping system for molecular epidemiological analysis of 369 M. tuberculosis isolates from 3 regions of Brazil, a high TB-burden country. Deligotyping, targeting 43 large sequence polymorphisms (LSPs), and the MIRU-VNTRplus identification database were used to assess phylogenetic predictions. High congruence between the different typing results consistently revealed the countrywide supremacy of the Latin-American-Mediterranean (LAM) lineage, comprised of three main branches. In addition to an already known RDRio branch, at least one other branch characterized by a phylogenetically informative LAM3 spoligo-signature seems to be globally distributed beyond Brazil. Nevertheless, by distinguishing 321 genotypes in this strain population, combined MIRU-VNTR typing and spoligotyping demonstrated the presence of multiple distinct clones. The use of 15 to 24 loci discriminated 21 to 25% more strains within the LAM lineage, compared to a restricted lineage-specific locus set suggested to be used after SNP analysis. Noteworthy, 23 of the 28 molecular clusters identified were exclusively composed of patient isolates from a same region, consistent with expected patterns of mostly local TB transmission. Standard MIRU-VNTR typing combined with spoligotyping can reveal epidemiologically meaningful clonal diversity behind a dominant M. tuberculosis strain lineage in a high TB-burden country and is useful to explore international phylogenetical ramifications.

• On the mutation rates of spoligotypes and variable numbers of tandem repeat loci of Mycobacterium tuberculosis.

  Authors: Philip Supply, Stefan Niemann, Thierry Wirth

  Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases. 12/2010; 11(2):251-2.

  Reasonable estimation of mutation rates of molecular markers has implications for molecular epidemiology and evolutionary studies. A recent report (Reyes and Tanaka, 2010) suggested mutation rateReasonable estimation of mutation rates of molecular markers has implications for molecular epidemiology and evolutionary studies. A recent report (Reyes and Tanaka, 2010) suggested mutation rate estimates for variable number of tandem repeat (VNTR) loci in Mycobacterium tuberculosis that are at least an order of magnitude higher than those previously reported (Grant et al., 2008; Wirth et al., 2008). We believe that this deviation can be explained by methodological and analytical problems, including the use of a IS6110 RFLP mutation rate reference that is too indeterminate for subsequently estimating mutation rates of VNTR markers, the utilisation of an infinite allele model unsuitable for the latter markers, and the inclusion of some hypervariable VNTR loci that were excluded from the previous comparison reports. Moreover, the ensuing conclusion on potential (general) underestimation of the extent of recent transmission when using VNTR markers (i.e. because they would mutate so rapidly) conflicts with the large amount of published data demonstrating only rare changes of standard VNTR loci among isolates from clonal transmission or infection.

• Microevolution of Mycobacterium tuberculosis in a tuberculosis patient.

  Authors: Sahal A M Al-Hajoj, Onno Akkerman, Ida Parwati, Saad al-Gamdi, Zeaur Rahim, Dick van Soolingen, Jakko van Ingen, Philip Supply, Adri G M van der Zanden

  Journal of clinical microbiology. 10/2010; 48(10):3813-6.

  Five Mycobacterium tuberculosis isolates were obtained from three body sites from a Dutch patient. The isolates displayed a single genotype by 24-locus MIRU-VNTR typing (except for a single locus notFive Mycobacterium tuberculosis isolates were obtained from three body sites from a Dutch patient. The isolates displayed a single genotype by 24-locus MIRU-VNTR typing (except for a single locus not amplified from one isolate) but were differentiated by small variations in IS6110 fingerprints, spoligotypes, 6 hypervariable MIRU-VNTR loci, and/or DiversiLab profiles, revealing patterns of microevolution in a clonal infection.

• MIRU-VNTRplus: a web tool for polyphasic genotyping of Mycobacterium tuberculosis complex bacteria.

  Authors: Thomas Weniger, Justina Krawczyk, Philip Supply, Stefan Niemann, Dag Harmsen

  Nucleic acids research. 05/2010; 38(Web Server issue):W326-31.

  Harmonized typing of bacteria and easy identification of locally or internationally circulating clones are essential for epidemiological surveillance and disease control. For MycobacteriumHarmonized typing of bacteria and easy identification of locally or internationally circulating clones are essential for epidemiological surveillance and disease control. For Mycobacterium tuberculosis complex (MTBC) species, multi-locus variable number tandem repeat analysis (MLVA) targeting mycobacterial interspersed repetitive units (MIRU) has been internationally adopted as the new standard, portable, reproducible and discriminatory typing method. However, no specialized bioinformatics web tools are available for analysing MLVA data in combination with other, complementary typing data. Therefore, we have developed the web application MIRU-VNTRplus (http://www.miru-vntrplus.org). This freely accessible service allows users to analyse genotyping data of their strains alone or in comparison with a reference database of strains representing the major MTBC lineages. Analysis and comparisons of genotypes can be based on MLVA-, spoligotype-, large sequence polymorphism and single nucleotide polymorphism data, or on a weighted combination of these markers. Tools for data exploration include search for similar strains, creation of phylogenetic and minimum spanning trees and mapping of geographic information. To facilitate scientific communication, an expanding genotype nomenclature (MLVA MtbC15-9 type) that can be queried via a web- or a SOAP-interface has been implemented. An extensive documentation guides users through all application functions.

• Determination of genotypic diversity of Mycobacterium avium subspecies from human and animal origins by mycobacterial interspersed repetitive-unit-variable-number tandem-repeat and IS1311 restriction fragment length polymorphism typing methods.

  Authors: Nicolas Radomski, Virginie C Thibault, Claudine Karoui, Krystel de Cruz, Thierry Cochard, Cristina Gutiérrez, Philip Supply, Frank Biet, María Laura Boschiroli

  Journal of clinical microbiology. 04/2010; 48(4):1026-34.

  Members of the Mycobacterium avium complex (MAC) are ubiquitous bacteria that can be found in water, food, and other environmental samples and are considered opportunistic pathogens for numerousMembers of the Mycobacterium avium complex (MAC) are ubiquitous bacteria that can be found in water, food, and other environmental samples and are considered opportunistic pathogens for numerous animal species, mainly birds and pigs, as well as for humans. We have recently demonstrated the usefulness of a PCR-based mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing for the molecular characterization of M. avium subsp. paratuberculosis and M. avium strains exclusively isolated from AIDS patients. In the present study we extended our analysis, based on eight MIRU-VNTR markers, to a strain collection comprehensively comprising the other M. avium subspecies, including M. avium subsp. avium, M. avium subsp. hominissuis, and M. avium subsp. silvaticum, isolated from numerous animal species, HIV-positive and HIV-negative humans, and environmental sources. All strains were fully typeable, with the discriminatory index being 0.885, which is almost equal to that obtained by IS1311 restriction fragment length polymorphism (RFLP) typing as a reference. In contrast to IS1311 RFLP typing, MIRU-VNTR typing was able to further discriminate M. avium subsp. avium strains. MIRU-VNTR alleles strongly associated with or specific for M. avium subspecies were detected in several markers. Moreover, the MIRU-VNTR typing-based results were consistent with a scenario of the independent evolution of M. avium subsp. avium/M. avium subsp. silvaticum and M. avium subsp. paratuberculosis from M. avium subsp. hominissuis, previously proposed on the basis of multilocus sequence analysis. MIRU-VNTR typing therefore appears to be a convenient typing method capable of distinguishing the three main subspecies and strains of the complex and providing new epidemiological knowledge on MAC.

• Impact of HIV infection on the recurrence of tuberculosis in South India.

  Authors: Sujatha Narayanan, Soumya Swaminathan, Philip Supply, Sivakumar Shanmugam, Gopalan Narendran, Lalitha Hari, Ranjani Ramachandran, Camille Locht, Mohideen Shaheed Jawahar, Paranji Raman Narayanan

  The Journal of infectious diseases. 03/2010; 201(5):691-703.

  There is limited information on the relative proportion of reactivation and reinfection at the time of recurrence among human immunodeficiency virus (HIV)-infected and HIV-uninfected patients who areThere is limited information on the relative proportion of reactivation and reinfection at the time of recurrence among human immunodeficiency virus (HIV)-infected and HIV-uninfected patients who are successfully treated for tuberculosis infection in India. HIV-infected and HIV-uninfected patients with sputum culture-positive pulmonary tuberculosis were treated with short-course regimens and followed up for 36 months at the Tuberculosis Research Centre, South India. Bacteriologic recurrences were documented, and typing of strains was performed using 3 different genotypic techniques: restriction fragment length polymorphism (RFLP) by IS6110, spoligotyping, and mycobacterial interspersed repeat unit (MIRU)-variable number tandem repeat (VNTR). DNA fingerprints of paired Mycobacterium tuberculosis isolates (baseline and recurrence) were compared. Among 44 HIV-infected and 30 HIV-uninfected patients with recurrent tuberculosis during the period July 1999 to October 2005, 25 and 23 paired isolates, respectively, were typed using all 3 methods. Recurrence was due to exogenous reinfection in 88% of HIV-infected and 9% of HIV-uninfected patients (P<.05). Among recurrent isolates, the HIV-infected patients showed more clustering, as well as a higher proportion of drug resistance, including multidrug resistance. In India, a tuberculosis-endemic country, most recurrences after successful treatment of tuberculosis are due to exogenous reinfection in HIV-infected persons and endogenous reactivation in HIV-uninfected persons. Strategies for prevention and treatment of tuberculosis infection must take these findings into consideration.

• MIRU-VNTR

  Authors: Thomas Weniger, Justina Krawczyk, Philip Supply, Stefan Niemann, Dag Harmsen

  Nucleic Acids Research. 01/2010; 38:326-331.

• Genetic diversity of Mycobacterium tuberculosis isolates from a tertiary tuberculosis hospital in South Korea.

  Authors: Isdore Chola Shamputa, Jongseok Lee, Caroline Allix-Béguec, Eun-Jin Cho, Ji-Im Lee, Vignesh Rajan, Eun Gae Lee, Jin Hong Min, Matthew W. Carroll, Lisa C Goldfeder, Jin Hee Kim, Hyung Seok Kang, Soohee Hwang, Seok-Yong Eum, Seung Kyu Park, Hyeyoung Lee, Philip Supply, Sang-Nae Cho, Laura E Via, Clifton E Barry

  Journal of clinical microbiology. 12/2009;

  Tuberculosis (TB) remains an immense public health problem in the Republic of Korea despite more than a five fold decrease in the prevalence of the disease over the last three decades. The rise inTuberculosis (TB) remains an immense public health problem in the Republic of Korea despite more than a five fold decrease in the prevalence of the disease over the last three decades. The rise in drug resistant TB has compounded the situation. We analyzed 208 clinical isolates of M. tuberculosis from the National Masan Tuberculosis Hospital by spoligotyping, IS6110-Restriction Fragment Length Polymorphism (RFLP) and 24-locus based mycobacterial interspersed repetitive units-variable number tandem repeats (MIRU-VNTR) to assess the diversity and transmission dynamics of the tubercle bacilli in the Republic of Korea. The majority of the isolates (97.1%) belonged to the Beijing genotype. Cluster analysis by MIRU-VNTR yielded a low clustering rate of 22.3% with most of the clusters comprising isolates with diverse drug resistance patterns. The discriminatory capacity of the typing methods was high for RFLP and MIRU-VNTR (h=0.99) but low for Spoligotyping (h=0.31). Although analysis of 19 MIRU-VNTR loci was needed to achieve maximum discrimination, an informative set of 8 loci (960, 1955, 2163b, 2165, 2996, 3192, 4052 and 4348) (h=0.98) that was able to differentiate most of the closely related strains was identified. These findings suggest that 24-locus based MIRU-VNTR typing is a likely suitable alternative to RFLP to differentiate clinical isolates in this setting dominated by M. tuberculosis Beijing strains. Within the study limits, our results also suggest that the problem of drug resistant TB in the Republic of Korea may be largely due to acquired resistance as opposed to transmission.

• Combined multilocus short sequence repeat and mycobacterial interspersed repetitive unit- variable-number tandem repeat typing of Mycobacterium avium subsp. paratuberculosis isolates.

  Authors: Virginie C Thibault, Maggy Grayon, Maria Laura Boschiroli, Eve Willery, Caroline Allix-Béguec, Karen Stevenson, Franck Biet, Philip Supply

  Journal of clinical microbiology. 11/2008;

  Short sequence repeat (SSR)-sequencing was applied to 127 Mycobacterium avium subsp. paratuberculosis isolates typed by mycobacterial interspersed repetitive unit-variable-number tandem repeatsShort sequence repeat (SSR)-sequencing was applied to 127 Mycobacterium avium subsp. paratuberculosis isolates typed by mycobacterial interspersed repetitive unit-variable-number tandem repeats (MIRU-VNTR) and IS900 restriction fragment length polymorphim (RFLP). Combined MIRU-VNTR and SSR typing followed by secondary IS900 RFLP typing is an improved approach to high-resolution genotyping of this pathogen.

• Origin, Spread and Demography of the Mycobacterium tuberculosis Complex.

  Authors: Thierry Wirth, Falk Hildebrand, Caroline Allix-Béguec, Florian Wölbeling, Tanja Kubica, Kristin Kremer, Dick van Soolingen, Sabine Rüsch-Gerdes, Camille Locht, Sylvain Brisse, Axel Meyer, Philip Supply, Stefan Niemann

  PLoS pathogens. 10/2008; 4(9):e1000160.

  The evolutionary timing and spread of the Mycobacterium tuberculosis complex (MTBC), one of the most successful groups of bacterial pathogens, remains largely unknown. Here, using mycobacterialThe evolutionary timing and spread of the Mycobacterium tuberculosis complex (MTBC), one of the most successful groups of bacterial pathogens, remains largely unknown. Here, using mycobacterial tandem repeat sequences as genetic markers, we show that the MTBC consists of two independent clades, one composed exclusively of M. tuberculosis lineages from humans and the other composed of both animal and human isolates. The latter also likely derived from a human pathogenic lineage, supporting the hypothesis of an original human host. Using Bayesian statistics and experimental data on the variability of the mycobacterial markers in infected patients, we estimated the age of the MTBC at 40,000 years, coinciding with the expansion of "modern" human populations out of Africa. Furthermore, coalescence analysis revealed a strong and recent demographic expansion in almost all M. tuberculosis lineages, which coincides with the human population explosion over the last two centuries. These findings thus unveil the dynamic dimension of the association between human host and pathogen populations.

• Evaluation and user-strategy of MIRU-VNTRplus, a multifunctional database for on-line analysis of genotyping data and phylogenetic identification of Mycobacterium tuberculosis complex isolates.

  Authors: Caroline Allix-Béguec, Dag Harmsen, Thomas Weniger, Philip Supply, Stefan Niemann

  Journal of clinical microbiology. 06/2008;

  Because of its portable data, discriminatory power, and recently proposed standardization, mycobacterial interspersed repetitive unit-variable number of tandem repeat (MIRU-VNTR) typing has become aBecause of its portable data, discriminatory power, and recently proposed standardization, mycobacterial interspersed repetitive unit-variable number of tandem repeat (MIRU-VNTR) typing has become a major method for epidemiological tracking of Mycobacterium tuberculosis complex (MTBC) clones. However, no public MIRU-VNTR database based on well-characterized reference strains was available hitherto for easy strain identification. Therefore, a collection of 186 reference strains representing the primary MTBC lineages was used to build up a database freely accessible at http://www.MIRU-VNTRplus.org. For each strain, geographical origin and drug-susceptibility profile was stored together with comprehensive genetic lineage information including 24-locus MIRU-VNTR profile, spoligotyping pattern, single nucleotide and large sequence polymorphism profile, and IS6110 restriction fragment length polymorphism fingerprint. Thanks to flexible import functions, single or multiple user strain(s) can be analyzed e.g. for lineage identification with or without using reference strains, by best-match or tree-based analyses employing single or combined marker datasets. Results can be easily exported. Here, we evaluated the database consistency and various analysis parameters, both by testing the reference collection against itself and by using an external population-based dataset comprising 629 different strains. In the optimal conditions found, lineage predictions based on 24-locus MIRU-VNTR-typing optionally combined with spoligotyping were verified in > 99% of the cases. Based on this evaluation, a user-strategy was defined, including best match analysis followed, if necessary, by tree-based analysis. The MIRU-VNTRplus database is a powerful tool for high-resolution clonal identification, which has little equivalent in terms of functionalities among the bacterial genotyping databases available so far.

• Three-year population-based evaluation of standardized mycobacterial interspersed repetitive-unit-variable-number tandem-repeat typing of Mycobacterium tuberculosis.

  Authors: Caroline Allix-Béguec, Maryse Fauville-Dufaux, Philip Supply

  Journal of clinical microbiology. 05/2008; 46(4):1398-406.

  Standardized mycobacterial interspersed repetitive-unit-variable-number tandem repeat (MIRU-VNTR) typing based on 15 and 24 loci recently has been proposed for Mycobacterium tuberculosis genotyping.Standardized mycobacterial interspersed repetitive-unit-variable-number tandem repeat (MIRU-VNTR) typing based on 15 and 24 loci recently has been proposed for Mycobacterium tuberculosis genotyping. So far, this optimized system has been assessed in a single, 1-year population-based study performed in Germany (M. C. Oelemann, R. Diel, V. Vatin, W. Haas, S. Rusch-Gerdes, C. Locht, S. Niemann, and P. Supply, J. Clin. Microbiol. 45:691-697, 2007). Here, we evaluated these optimized formats in a much larger population-based study conducted during 39 months in the Brussels capital region of Belgium. Isolates from 807 patients were genotyped. The resolution power, cluster, and lineage identification by the standardized MIRU-VNTR sets were compared to those obtained using standardized IS6110-restriction fragment length polymorphism (RFLP), spoligotyping, and a previous 12-MIRU-VNTR-locus set. On a subset representing 77% of the cases during a 16-month period, a high concordance was observed between unique isolates or strain clusters as defined by standardized MIRU-VNTR and IS6110-RFLP (i.e., more than five IS6110 bands). When extended to the entire population-based collection, the discriminatory subset of 15 loci decreased the strain-clustering rate by almost twofold compared to that of the old 12-locus set. The addition of the nine ancillary MIRU-VNTR loci and/or spoligotyping only slightly further decreased this strain-clustering rate. Familial, social, and/or geographic proximity links were found in 48% of the clusters identified, and well-known risk factors for tuberculosis transmission were identified. Finally, an excellent correspondence was determined between our MIRU-VNTR-spoligotyping strain identifications and external reference strain lineages included in the MIRU-VNTRplus database and identified by, e.g., large sequence polymorphisms. Our results reinforce the proposal of standardized MIRU-VNTR typing as a new reference genotyping method for the epidemiological and phylogenetic screening of M. tuberculosis strains.

• New variable-number tandem-repeat markers for typing Mycobacterium avium subsp. paratuberculosis and M. avium strains: comparison with IS900 and IS1245 restriction fragment length polymorphism typing.

  Authors: Virginie C Thibault, Maggy Grayon, Maria Laura Boschiroli, Christine Hubbans, Pieter Overduin, Karen Stevenson, Maria Cristina Gutierrez, Philip Supply, Franck Biet

  Journal of clinical microbiology. 09/2007; 45(8):2404-10.

  Mycobacterium avium subsp. paratuberculosis, the etiological agent of paratuberculosis, affects a wide range of domestic ruminants and has been suggested to be involved in Crohn's disease in humans.Mycobacterium avium subsp. paratuberculosis, the etiological agent of paratuberculosis, affects a wide range of domestic ruminants and has been suggested to be involved in Crohn's disease in humans. Most available methods for identifying and differentiating strains of this difficult species are technically demanding and have limited discriminatory power. Here, we report the identification of novel PCR-based typing markers consisting of variable-number tandem repeats (VNTRs) of genetic elements called mycobacterial interspersed repetitive units (MIRUs). Eight markers were applied to 183 M. avium subsp. paratuberculosis isolates from bovine, caprine, ovine, cervine, leporine, and human origins from 10 different countries and to 82 human isolates of the closely related species M. avium from France. Among the M. avium subsp. paratuberculosis isolates, 21 patterns were found by MIRU-VNTR typing, with a discriminatory index of 0.751. The predominant R01 IS900 restriction fragment length polymorphism type, comprising 131 isolates, was divided into 15 MIRU-VNTR types. Among the 82 M. avium isolates, the eight MIRU-VNTR loci distinguished 30 types, none of which was shared by M. avium subsp. paratuberculosis isolates, resulting in a discriminatory index of 0.889. Our results suggest that MIRU-VNTR typing is a fast typing method that, in combination with other methods, might prove to be optimal for PCR-based molecular epidemiological studies of M. avium/M. avium subsp. paratuberculosis pathogens. In addition, presumably identical M. avium subsp. paratuberculosis 316F vaccine strains originating from the Weybridge laboratory and from different commercial batches from Mérial actually differed by one or both typing methods. These results indicate a substantial degree of genetic drift among different vaccine preparations, which has important implications for prophylactic approaches.

• Assessment of an optimized mycobacterial interspersed repetitive- unit-variable-number tandem-repeat typing system combined with spoligotyping for population-based molecular epidemiology studies of tuberculosis.

  Authors: Mara Cardoso Oelemann, Roland Diel, Vincent Vatin, Walter Haas, Sabine Rüsch-Gerdes, Camille Locht, Stefan Niemann, Philip Supply

  Journal of clinical microbiology. 03/2007; 45(3):691-7.

  An optimized set of 24 mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) loci, including a discriminatory subset of 15 loci, has recently been defined for theAn optimized set of 24 mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) loci, including a discriminatory subset of 15 loci, has recently been defined for the typing of Mycobacterium tuberculosis. Here, we evaluated the performances of this MIRU-VNTR typing system in combination with spoligotyping for the detection of transmission chains in a population-based study comprising 91% of culture-confirmed tuberculosis patients reported in 2003 in Hamburg, Germany. Of the 154 isolates investigated, more than 90% had high IS6110 copy numbers (>/=6). IS6110 restriction fragment length polymorphism (RFLP) typing resulted in 13 clusters, 5 of which had a confirmed epidemiological link. All five, as well as six of the eight IS6110 clusters with no identified epidemiological link, were perfectly matched by MIRU-VNTR typing with the 24 loci. Two IS6110 clusters were split by differences into 6 to 12 MIRU-VNTR loci, clearly supporting the absence of a link, as judged by contact tracing data. In contrast, only one MIRU-VNTR cluster, grouping what were probably epidemiologically unlinked isolates, was split by IS6110 RFLP. However, these isolates were also distinguished by spoligotyping. Both the optimized 24-locus and 15-locus sets thus showed a comparable to slightly better predictive value, especially when combined with spoligotyping, than the current gold standard IS6110 RFLP for the study of tuberculosis transmission in Hamburg. Because the epidemiological characteristics of this setting are similar to those of many developed countries, these results support the wide applicability of this real-time genotyping approach for population-based studies of M. tuberculosis transmission.

• Proposal for standardization of optimized mycobacterial interspersed repetitive unit-variable-number tandem repeat typing of Mycobacterium tuberculosis.

  Authors: Philip Supply, Caroline Allix, Sarah Lesjean, Mara Cardoso Oelemann, Sabine Rüsch-Gerdes, Eve Willery, Evgueni Savine, Petra de Haas, Henk van Deutekom, Solvig Roring [......] Christophe Sola, Nalin Rastogi, Vincent Vatin, Maria Cristina Gutierrez, Maryse Fauville, Stefan Niemann, Robin Skuce, Kristin Kremer, Camille Locht, Dick van Soolingen

  Journal of clinical microbiology. 01/2007; 44(12):4498-510.

  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 theMolecular 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.

• Predominance of ancestral lineages of Mycobacterium tuberculosis in India.

  Authors: M Cristina Gutierrez, Niyaz Ahmed, Eve Willery, Sujatha Narayanan, Seyed E Hasnain, Devendra S Chauhan, Vishwa M Katoch, Véronique Vincent, Camille Locht, Philip Supply

  Emerging infectious diseases. 10/2006; 12(9):1367-74.

  Although India has the highest prevalence of tuberculosis (TB) worldwide, the genetic diversity of Mycobacterium tuberculosis in India is largely unknown. A collection of 91 isolates originating fromAlthough India has the highest prevalence of tuberculosis (TB) worldwide, the genetic diversity of Mycobacterium tuberculosis in India is largely unknown. A collection of 91 isolates originating from 12 different regions spread across the country were analyzed by genotyping using 21 loci with variable-number tandem repeats (VNTRs), by spoligotyping, by principal genetic grouping (PGG), and by deletion analysis of M. tuberculosis-specific deletion region 1. The isolates showed highly diverse VNTR genotypes. Nevertheless, highly congruent groupings identified by using the 4 independent sets of markers permitted a clear definition of 3 prevalent PGG1 lineages, which corresponded to the "ancestral" East African-Indian, the Delhi, and the Beijing/W genogroups. A few isolates from PGG2 lineages and a single representative of the presumably most recent PGG3 were identified. These observations suggest a predominance of ancestral M. tuberculosis genotypes in the Indian subcontinent, which supports the hypothesis that India is an ancient endemic focus of TB.