Biomedical subjects

Kristin Kremer

Publications and source records attributed to Kristin Kremer.

At least 19 recordsLinked to original sources

PubMed2010

Human T cell epitopes of Mycobacterium tuberculosis are evolutionarily hyperconserved.

Mycobacterium tuberculosis is an obligate human pathogen capable of persisting in individual hosts for decades. We sequenced the genomes of 21 strains representative of the global diversity and six major lineages of the M. tuberculosis complex (MTBC) at 40- to 90-fold coverage using Illumina next-generation DNA sequencing. We constructed a genome-wide phylogeny based on these genome sequences. Comparative analyses of the sequences showed, as expected, that essential genes in MTBC were more evolutionarily conserved than nonessential genes. Notably, however, most of the 491 experimentally confirmed human T cell epitopes showed little sequence variation and had a lower ratio of nonsynonymous to synonymous changes than seen in essential and nonessential genes. We confirmed these findings in an additional data set consisting of 16 antigens in 99 MTBC strains. These findings are consistent with strong purifying selection acting on these epitopes, implying that MTBC might benefit from recognition by human T cells.

Antigens, Bacterial↗

PubMed2006

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

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.

Bacterial Typing Techniques↗

PubMed2006

Beijing genotype of Mycobacterium tuberculosis is significantly associated with human immunodeficiency virus infection and multidrug resistance in cases of tuberculous meningitis.

Multidrug-resistant tuberculous meningitis is fatal without rapid diagnosis and use of second-line therapy. It is more common in human immunodeficiency virus (HIV)-positive patients. Beijing genotype strains of Mycobacterium tuberculosis are associated with drug resistance, particularly multidrug resistance, and their prevalence is increasing worldwide. The prevalence of Beijing genotype strains among Mycobacterium tuberculosis isolates from the cerebrospinal fluid of HIV-positive (n = 35) and HIV-negative (n = 187) patients in Ho Chi Minh City was determined. The Beijing genotype was significantly associated with HIV status (odds ratio [OR] = 2.95 [95% confidence interval {CI}, 1.38 to 6.44]; P = 0.016), resistance to any drug (OR = 3.34 [95% CI, 1.87 to 5.95]; P < 0.001) and multidrug resistance (Fisher's exact test; P = 0.001). The association of the Beijing genotype with drug resistance was independent of HIV status. This is the first report of Beijing genotype association with HIV status, which may be an association unique to tuberculous meningitis.

Adolescent↗

PubMed2006

Mycobacterium tuberculosis complex genetic diversity: mining the fourth international spoligotyping database (SpolDB4) for classification, population genetics and epidemiology.

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.

Computational Biology↗

PubMed2006

Variable host-pathogen compatibility in Mycobacterium tuberculosis.

Mycobacterium tuberculosis remains a major cause of morbidity and mortality worldwide. Studies have reported human pathogens to have geographically structured population genetics, some of which have been linked to ancient human migrations. However, no study has addressed the potential evolutionary consequences of such longstanding human-pathogen associations. Here, we demonstrate that the global population structure of M. tuberculosis is defined by six phylogeographical lineages, each associated with specific, sympatric human populations. In an urban cosmopolitan environment, mycobacterial lineages were much more likely to spread in sympatric than in allopatric patient populations. Tuberculosis cases that did occur in allopatric hosts disproportionately involved high-risk individuals with impaired host resistance. These observations suggest that mycobacterial lineages are adapted to particular human populations. If confirmed, our findings have important implications for tuberculosis control and vaccine development.

Adaptation, Physiological↗

PubMed2005

Ecotypes of the Mycobacterium tuberculosis complex.

A phylogeny of the Mycobacterium tuberculosis complex has recently shown that the animal-adapted strains are found in a single lineage marked by the deletion of chromosomal region 9 (RD9) [Brosch et al., 2002. A new evolutionary scenario for the Mycobacterium tuberculosis complex. Proc. Natl Acad. Sci. USA 99 (6), 3684-3689]. We have obtained the spoligotype patterns of the RD9 deleted strains used to generate this new evolutionary scenario and we show that the presence of spoligotype spacers 3, 9, 16, 39, and 40-43 is phylogenetically informative in this lineage. We have used the phylogenetically informative spoligotype spacers to screen a database of spoligotype patterns and have identified further members of a group of strains apparently host-adapted to antelopes. The presence of the spoligotype spacers is congruent with the phylogeny generated by chromosomal deletions, suggesting that recombination is rare or absent between strains of this lineage. The phylogenetically informative spacers, in concert with the previously identified single nucleotide mutations and chromosomal deletions, can be used to identify a series of clades in the RD9 deleted lineage each with a separate host preference. Finally, we discuss the application of the ecotype concept to this series of clades and suggest that the M. tuberculosis complex may best be described as a series of host-adapted ecotypes.

Animals↗

PubMed2005

Revisiting the evolution of Mycobacterium bovis.

Though careful consideration has been placed towards genetic characterization of tubercle bacillus isolates causing disease in humans, those causing disease predominantly among wild and domesticated mammals have received less attention. In contrast to Mycobacterium tuberculosis, whose host range is largely specific to humans, M. bovis and "M bovis-like" organisms infect a broad range of animal species beyond their most prominent host in cattle. To determine whether strains of variable genomic content are associated with distinct distributions of disease, the DNA contents of M. bovis or M. bovis-like isolates from a variety of hosts were investigated via Affymetrix GeneChip. Consistent with previous genomic analysis of the M. tuberculosis complex (MTC), large sequence polymorphisms of putative diagnostic and biological consequence were able to unambiguously distinguish interrogated isolates. The distribution of deleted regions indicates organisms genomically removed from M. bovis and also points to structured genomic variability within M. bovis. Certain genomic profiles spanned a variety of hosts but were clustered by geography, while others associated primarily with host type. In contrast to the prevailing assumption that M. bovis has broad host capacity, genomic profiles suggest that distinct MTC lineages differentially infect a variety of mammals. From this, a phylogenetic stratification of genotypes offers a predictive framework upon which to base future genetic and phenotypic studies of the MTC.

Evolution, Molecular↗

PubMed2005

Utility of mycobacterial interspersed repetitive unit typing for differentiating multidrug-resistant Mycobacterium tuberculosis isolates of the Beijing family.

Mycobacterial interspersed repetitive unit (MIRU) typing has been found to allow rapid, reliable, high-throughput genotyping of Mycobacterium tuberculosis and may represent a feasible approach to study global M. tuberculosis molecular epidemiology. To evaluate the use of MIRU typing in discriminating drug-resistant M. tuberculosis strains of the Beijing genotype family, 102 multidrug-resistant (MDR) clinical isolates and 253 randomly selected non-MDR isolates collected from 2000 to 2003 in Hong Kong were subjected to 12-locus MIRU typing, spoligotyping, and IS6110 restriction fragment length polymorphism (RFLP) typing. Spoligotyping showed that 243 (68.5%) of 355 isolates belonged to Beijing family genotype. MIRU typing showed lower discrimination in differentiating between the Beijing family strains (Hunter-Gaston discriminative index [HGI] of 0.8827) compared with the IS6110 RFLP method (HGI = 0.9979). For non-Beijing strains, MIRU typing provided discrimination (HGI = 0.9929) comparable to that of the RFLP method (HGI = 0.9961). There was no remarkable difference in discrimination power between the two methods in differentiating both within and between MDR and non-MDR strains of M. tuberculosis. Dendrograms constructed with the MIRU typing data showed a clear segregation between the Beijing and non-Beijing genotype. Addition of RFLP to MIRU typing offered a higher discrimination ability (92.6%) than did addition of MIRU typing to RFLP (40.0%). This supported the potential use of this method to analyze the global genetic diversity of MDR M. tuberculosis strains that may be at different levels of evolutionary divergence.

Bacterial Typing Techniques↗

PubMed2005

Use of variable-number tandem-repeat typing to differentiate Mycobacterium tuberculosis Beijing family isolates from Hong Kong and comparison with IS6110 restriction fragment length polymorphism typing and spoligotyping.

The Mycobacterium tuberculosis Beijing family isolates may cause more than a quarter of all tuberculosis cases worldwide, are emerging in some areas, and are often associated with drug resistance. Early recognition of transmission of this genotype is therefore important. To evaluate the usefulness of variable-number tandem-repeat (VNTR) typing to discriminate and recognize strains of the Beijing family, M. tuberculosis isolates from Hong Kong were subjected to VNTR analysis, spoligotyping, and IS6110 restriction fragment length polymorphism (RFLP) typing. The allelic diversity of the 14 VNTR loci included in the analysis varied from 0 to 0.618 among Beijing strains. The discriminatory power of VNTR analysis was slightly lower than that of IS6110 RFLP. Our analysis shows that VNTR typing, which has many practical advantages over RFLP typing, can be used for epidemiological studies of Beijing strains. However, VNTR-defined clusters should be subtyped with IS6110 RFLP for maximal resolution.

Bacterial Typing Techniques↗

PubMed2005

Fast ligation-mediated PCR, a fast and reliable method for IS6110-based typing of Mycobacterium tuberculosis complex.

IS6110 restriction fragment length polymorphism (RFLP) analysis is the most widely applied method for strain differentiation of Mycobacterium tuberculosis complex. We have previously described mixed-linker PCR, an IS6110-based PCR method that favorably compared with other typing methods for M. tuberculosis complex according to reproducibility and ability to differentiate between strains. Here we report the further development of this method, called fast ligation-mediated PCR (FLiP), which allows analysis of strains within one working day and starting from less than 1 ng of mycobacterial DNA or a crude cell lysate. Blinded analysis of a standard set of 131 M. tuberculosis complex and nontuberculous isolates showed the ability to differentiate 81 types among 90 M. tuberculosis complex isolates with 84 different IS6110 RFLP fingerprint patterns and detected 97% of the 31 duplicate samples. We suggest that FLiP can serve to rapidly detect chains of transmission prior to starting high-throughput analysis or standard IS6110 RFLP. It may as well serve as a secondary typing technique for other, non-IS6110-based methods.

Bacterial Typing Techniques↗

PubMed2005

Discriminatory power and reproducibility of novel DNA typing methods for Mycobacterium tuberculosis complex strains.

In recent years various novel DNA typing methods have been developed which are faster and easier to perform than the current internationally standardized IS6110 restriction fragment length polymorphism typing method. However, there has been no overview of the utility of these novel typing methods, and it is largely unknown how they compare to previously published methods. In this study, the discriminative power and reproducibility of nine recently described PCR-based typing methods for Mycobacterium tuberculosis were investigated using the strain collection of the interlaboratory study of Kremer et al. This strain collection contains 90 M. tuberculosis complex and 10 non-M. tuberculosis complex mycobacterial strains, as well as 31 duplicated DNA samples to assess reproducibility. The highest reproducibility was found with variable numbers of tandem repeat typing using mycobacterial interspersed repetitive units (MIRU VNTR) and fast ligation-mediated PCR (FLiP), followed by second-generation spoligotyping, ligation-mediated PCR (LM-PCR), VNTR typing using five repeat loci identified at the Queens University of Belfast (QUB VNTR), and the Amadio speciation PCR. Poor reproducibility was associated with fluorescent amplified fragment length polymorphism typing, which was performed in three different laboratories. The methods were ordered from highest discrimination to lowest by the Hunter-Gaston discriminative index as follows: QUB VNTR typing, MIRU VNTR typing, FLiP, LM-PCR, and spoligotyping. We conclude that both VNTR typing methods and FLiP typing are rapid, highly reliable, and discriminative epidemiological typing methods for M. tuberculosis and that VNTR typing is the epidemiological typing method of choice for the near future.

Bacterial Typing Techniques↗

PubMed2005

Targeted hybridization of IS6110 fingerprints identifies the W-Beijing Mycobacterium tuberculosis strains among clinical isolates.

Targeted IS6110-based RFLP genotyping can be applied to rapidly identify specific groups of biomedically/epidemiologically relevant Mycobacterium tuberculosis clinical isolates. One such group is the W-Beijing strain family (also known as Beijing/W), implicated in significant nosocomial and community outbreaks worldwide. Using previously defined criteria, we developed a simple and accurate method to identify members of the W-Beijing family, based on rehybridization of Southern blot membranes used previously in routine IS6110 DNA fingerprint analysis. The hybridization probe constructed ("W-Beijing polyprobe") contains the PCR-amplified fragments specific for three M. tuberculosis chromosomal loci used for the identification of W-Beijing strains. The targets include the dnaA-dnaN and NTF regions and the direct repeat locus. A total of 526 selected clinical isolates (representative of 253 different IS6110-defined strain types) were analyzed using the W-Beijing polyprobe. A total of 148 isolates from this collection were found to be members of the W-Beijing phylogenetic lineage, comprising 106 strains from the W-Beijing family (46 clusters) and 42 related isolates. Rehybridization results were confirmed by computer-assisted analysis. The sensitivity and specificity of this method were estimated at 98.7% and 99.7%, respectively. This study demonstrates that the W-Beijing polyprobe can accurately and reliably discriminate members of the W-Beijing phylogenetic lineage and the W-Beijing family of M. tuberculosis strains.

Base Sequence↗

PubMed2005

Tuberculosis elimination in the Netherlands.

This study assessed progress towards tuberculosis (TB) elimination in the Netherlands by using DNA fingerprinting. Mycobacterium tuberculosis strains were defined as new if the IS6110 restriction fragment length polymorphism pattern had not been observed in any other patient during the previous 2 years. Other cases were defined as clustered and attributed to recent transmission. In the period 1995-2002, the incidence of TB with new strains was stable among non-Dutch residents and declined among the Dutch. However, the decline among the Dutch was restricted to those >or=65 years of age. Moreover, the average number of secondary cases per new strain did not change significantly over time. We conclude that the decline of TB in the Netherlands over the past decade was mainly the result of a cohort effect: older birth cohorts with high infection prevalence were replaced by those with lower infection prevalence. Under current epidemiologic conditions and control efforts, TB may not be eliminated.

Adult↗

PubMed2004

Mycobacterium tuberculosis, Beijing genotype strains not associated with radiological presentation of pulmonary tuberculosis.

Mycobacterium tuberculosis strains of the Beijing genotype have been involved in various outbreaks of multidrug-resistant tuberculosis. Some studies suggest that the infection with the Beijing genotype is associated with a different host immune response. Since this might also lead to a different chest X-ray (CXR) presentation, we compared CXRs of patients with pulmonary tuberculosis, 33 of whom were infected with the Beijing genotype and 76 with other genotypes. No significant differences were detected. We conclude that the Beijing genotype is not associated with a different CXR presentation.

Adult↗

PubMed2004

Genome structure in the vole bacillus, Mycobacterium microti, a member of the Mycobacterium tuberculosis complex with a low virulence for humans.

Mycobacterium microti, a member of the Mycobacterium tuberculosis complex, is phylogenetically closely related to M. tuberculosis, differing in a few biochemical properties. However, these species have different levels of virulence in different hosts; most notably M. microti shows lower virulence for humans than M. tuberculosis. This report presents genomic comparisons using DNA microarray analysis for an extensive study of the diversity of M. microti strains. Compared to M. tuberculosis H37Rv, 13 deletions were identified in 12 strains of M. microti, including the regions RD1 to RD10, which are also missing in Mycobacterium bovis BCG. In addition, four new deleted regions, named MiD1, RD1beta, MiD2 and MiD3, were identified. DNA sequencing was used to define the extent of most of the deletions in one strain. Although RD1 of M. bovis BCG and M. microti is thought to be crucial for attenuation, in this study, three of the four M. microti strains that were isolated from immunocompetent patients had the RD1 deletion. In fact, only the RD3 deletion was present in all of the strains examined, although deletions RD7, RD8 and MiD1 were found in almost all the M. microti strains. These deletions might therefore have some relation to the different host range of M. microti. It was also noticeable that of the 12 strains studied, only three were identical; these strains were all isolated from immunocompetent humans, suggesting that they could have arisen from a single source. Thus, this study shows that it is difficult to ascribe virulence to any particular pattern of deletion in M. microti.

Animals↗

PubMed2004

Characterization of ancestral Mycobacterium tuberculosis by multiple genetic markers and proposal of genotyping strategy.

Sixty-eight ancestral Mycobacterium tuberculosis isolates were previously identified by using the tuberculosis-specific deletion 1 (TbD1) PCR and mycobacterial interspersed-repetitive-unit-variable-number tandem repeat (MIRU-VNTR) typing (Y. J. Sun, R. Bellamy, A. S. G. Lee, S. T. Ng, S. Ravindran, S.-Y. Wong, C. Locht, P. Supply, and N. I. Paton, J. Clin. Microbiol. 42:1986-1993, 2004). These TbD1(+) ancestral isolates were further characterized and typed in this study by IS6110 restriction fragment length polymorphism (RFLP) typing, VNTR typing using exact tandem repeats (VNTR-ETR), and spoligotyping of the direct-repeat region. To our knowledge, this is the first characterization of this genogroup by multiple genetic markers based on a fairly large sample size. In this genogroup, all spoligotypes were characterized by the absence of spacers 29 to 32 and 34. In addition, VNTR-ETR typing could add further resolution to the clustered isolates identified by MIRU-VNTR, and the combination of MIRU-VNTR and VNTR-ETR, called MIRU-ETR, showed the highest discriminatory power for these strains compared to IS6110 RFLP typing and spoligotyping alone. However, MIRU-ETR appeared to still cluster some probably epidemiologically unrelated strains, as judged by IS6110 RFLP divergence. Therefore, a typing strategy based on stepwise combination of MIRU-ETR and IS6110 RFLP is proposed to achieve maximal discrimination for unrelated TbD1(+) strains. This typing strategy may be useful in areas where TbD1(+) ancestral strains are prevalent.

Bacterial Typing Techniques↗

PubMed2004

Genomic analysis distinguishes Mycobacterium africanum.

Mycobacterium africanum is thought to comprise a unique species within the Mycobacterium tuberculosis complex. M. africanum has traditionally been identified by phenotypic criteria, occupying an intermediate position between M. tuberculosis and M. bovis according to biochemical characteristics. Although M. africanum isolates present near-identical sequence homology to other species of the M. tuberculosis complex, several studies have uncovered large genomic regions variably deleted from certain M. africanum isolates. To further investigate the genomic characteristics of organisms characterized as M. africanum, the DNA content of 12 isolates was interrogated by using Affymetrix GeneChip. Analysis revealed genomic regions of M. tuberculosis deleted from all isolates of putative diagnostic and biological consequence. The distribution of deleted sequences suggests that M. africanum subtype II isolates are situated among strains of "modern" M. tuberculosis. In contrast, other M. africanum isolates (subtype I) constitute two distinct evolutionary branches within the M. tuberculosis complex. To test for an association between deleted sequences and biochemical attributes used for speciation, a phenotypically diverse panel of "M. africanum-like" isolates from Guinea-Bissau was tested for these deletions. These isolates clustered together within one of the M. africanum subtype I branches, irrespective of phenotype. These results indicate that convergent biochemical profiles can be independently obtained for M. tuberculosis complex members, challenging the traditional approach to M. tuberculosis complex speciation. Furthermore, the genomic results suggest a rational framework for defining M. africanum and provide tools to accurately assess its prevalence in clinical specimens.

Genome, Bacterial↗

PubMed2004

Definition of the Beijing/W lineage of Mycobacterium tuberculosis on the basis of genetic markers.

Mycobacterium tuberculosis Beijing genotype strains are highly prevalent in Asian countries and in the territory of the former Soviet Union. They are increasingly reported in other areas of the world and are frequently associated with tuberculosis outbreaks and drug resistance. Beijing genotype strains, including W strains, have been characterized by their highly similar multicopy IS6110 restriction fragment length polymorphism (RFLP) patterns, deletion of spacers 1 to 34 in the direct repeat region (Beijing spoligotype), and insertion of IS6110 in the genomic dnaA-dnaN locus. In this study the suitability and comparability of these three genetic markers to identify members of the Beijing lineage were evaluated. In a well-characterized collection of 1,020 M. tuberculosis isolates representative of the IS6110 RFLP genotypes found in The Netherlands, strains of two clades had spoligotypes characteristic of the Beijing lineage. A set of 19 Beijing reference RFLP patterns was selected to retrieve all Beijing strains from the Dutch database. These reference patterns gave a sensitivity of 98.1% and a specificity of 99.7% for identifying Beijing strains (defined by spoligotyping) in an international database of 1,084 strains. The usefulness of the reference patterns was also assessed with large DNA fingerprint databases in two other European countries and for identification strains from the W lineage found in the United States. A standardized definition for the identification of M. tuberculosis strains belonging to the Beijing/W lineage, as described in this work, will facilitate further studies on the spread and characterization of this widespread genotype family of M. tuberculosis strains.

China↗