Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology.
01 Apr 1997-Journal of Clinical Microbiology (American Society for Microbiology)-Vol. 35, Iss: 4, pp 907-914
TL;DR: A novel method based on strain-dependent hybridization patterns of in vitro-amplified DNA with multiple spacer oligonucleotides was found to differentiate M. bovis from M. tuberculosis, a distinction which is often difficult to make by traditional methods.
Abstract: Widespread use of DNA restriction fragment length polymorphism (RFLP) to differentiate strains of Mycobacterium tuberculosis to monitor the transmission of tuberculosis has been hampered by the need to culture this slow-growing organism and by the level of technical sophistication needed for RFLP typing. We have developed a simple method which allows simultaneous detection and typing of M. tuberculosis in clinical specimens and reduces the time between suspicion of the disease and typing from 1 or several months to 1 or 3 days. The method is based on polymorphism of the chromosomal DR locus, which contains a variable number of short direct repeats interspersed with nonrepetitive spacers. The method is referred to as spacer oligotyping or "spoligotyping" because it is based on strain-dependent hybridization patterns of in vitro-amplified DNA with multiple spacer oligonucleotides. Most of the clinical isolates tested showed unique hybridization patterns, whereas outbreak strains shared the same spoligotype. The types obtained from direct examination of clinical samples were identical to those obtained by using DNA from cultured M. tuberculosis. This novel preliminary study shows that the novel method may be a useful tool for rapid disclosure of linked outbreak cases in a community, in hospitals, or in other institutions and for monitoring of transmission of multidrug-resistant M. tuberculosis. Unexpectedly, spoligotyping was found to differentiate M. bovis from M. tuberculosis, a distinction which is often difficult to make by traditional methods.
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TL;DR: CRISPRFinder is described, a web service offering tools to detect CRISPRs including the shortest ones including one or two motifs, define DRs and extract spacers, and get the flanking sequences to determine the leader.
Abstract: Clustered regularly interspaced short palindromic repeats (CRISPRs) constitute a particular family of tandem repeats found in a wide range of prokaryotic genomes (half of eubacteria and almost all archaea). They consist of a succession of highly conserved regions (DR) varying in size from 23 to 47 bp, separated by similarly sized unique sequences (spacer) of usually viral origin. A CRISPR cluster is flanked on one side by an AT-rich sequence called the leader and assumed to be a transcriptional promoter. Recent studies suggest that this structure represents a putative RNA-interference-based immune system. Here we describe CRISPRFinder, a web service offering tools to (i) detect CRISPRs including the shortest ones (one or two motifs); (ii) define DRs and extract spacers; (iii) get the flanking sequences to determine the leader; (iv) blast spacers against Genbank database and (v) check if the DR is found elsewhere in prokaryotic sequenced genomes. CRISPRFinder is freely accessible at http://crispr.u-psud.fr/Server/CRISPRfinder.php.
1,689 citations
Cites background from "Simultaneous detection and strain d..."
...This strain-dependent polymorphism is especially interesting for epidemiological and phylogenetic studies (30,31)....
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TL;DR: A novel family of repetitive DNA sequences that is present among both domains of the prokaryotes but absent from eukaryotes or viruses is studied, characterized by direct repeats, varying in size from 21 to 37 bp, interspaced by similarly sized non‐repetitive sequences.
Abstract: Using in silico analysis we studied a novel family of repetitive DNA sequences that is present among both domains of the prokaryotes (Archaea and Bacteria), but absent from eukaryotes or viruses. This family is characterized by direct repeats, varying in size from 21 to 37 bp, interspaced by similarly sized non-repetitive sequences. To appreciate their characteri-stic structure, we will refer to this family as the clustered regularly interspaced short palindromic repeats (CRISPR). In most species with two or more CRISPR loci, these loci were flanked on one side by a common leader sequence of 300-500 b. The direct repeats and the leader sequences were conserved within a species, but dissimilar between species. The presence of multiple chromosomal CRISPR loci suggests that CRISPRs are mobile elements. Four CRISPR-associated (cas) genes were identified in CRISPR-containing prokaryotes that were absent from CRISPR-negative prokaryotes. The cas genes were invariably located adjacent to a CRISPR locus, indicating that the cas genes and CRISPR loci have a functional relationship. The cas3 gene showed motifs characteristic for helicases of the superfamily 2, and the cas4 gene showed motifs of the RecB family of exonucleases, suggesting that these genes are involved in DNA metabolism or gene expression. The spatial coherence of CRISPR and cas genes may stimulate new research on the genesis and biological role of these repeats and genes.
1,639 citations
Cites background or result from "Simultaneous detection and strain d..."
...pyogenes (Kamerbeek et al., 1997; Fang et al., 1998; Hoe et al., 1999), and preliminary investigations indicate that similar polymorphisms exist in other species, such as E....
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...A high degree of strain-dependent DNA polymorphism within the CRISPR loci has been observed in M. tuberculosis and in S. pyogenes (Kamerbeek et al., 1997; Fang et al., 1998; Hoe et al., 1999), and preliminary investigations indicate that similar polymorphisms exist in other species, such as E. coli…...
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...The CRISPR loci in clinical isolates of M. tuberculosis and S. pyogenes are extremely polymorphic, and this strain-dependent polymorphism has been exploited for epidemiological and taxonomic purposes (Kamerbeek et al., 1997; Hoe et al., 1999)....
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...tuberculosis, are very dissimilar (Mojica et al., 1995; 2000; Kamerbeek et al., 1997)....
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...This was consistent with the observation that CRISPR sequences in different species, such as E. coli and M. tuberculosis, are very dissimilar (Mojica et al., 1995; 2000; Kamerbeek et al., 1997)....
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TL;DR: The authors suggest that the spacer elements are the traces of past invasions by extrachromosomal elements, and hypothesize that they provide the cell immunity against phage infection, and more generally foreign DNA expression, by coding an anti-sense RNA.
Abstract: Numerous prokaryote genomes contain structures known as clustered regularly interspaced short palindromic repeats (CRISPRs), composed of 25-50 bp repeats separated by unique sequence spacers of similar length. CRISPR structures are found in the vicinity of four genes named cas1 to cas4. In silico analysis revealed another cluster of three genes associated with CRISPR structures in many bacterial species, named here as cas1B, cas5 and cas6, and also revealed a certain number of spacers that have homology with extant genes, most frequently derived from phages, but also derived from other extrachromosomal elements. Sequence analysis of CRISPR structures from 24 strains of Streptococcus thermophilus and Streptococcus vestibularis confirmed the homology of spacers with extrachromosomal elements. Phage sensitivity of S. thermophilus strains appears to be correlated with the number of spacers in the CRISPR locus the strain carries. The authors suggest that the spacer elements are the traces of past invasions by extrachromosomal elements, and hypothesize that they provide the cell immunity against phage infection, and more generally foreign DNA expression, by coding an anti-sense RNA. The presence of gene fragments in CRISPR structures and the nuclease motifs in cas genes of both cluster types suggests that CRISPR formation involves a DNA degradation step.
1,344 citations
TL;DR: A discriminatory subset of 15 loci with the highest evolutionary rates was defined that concentrated 96% of the total resolution obtained with the full 24-locus set, and its predictive value for evaluating M. tuberculosis transmission was found to be equal to that of IS6110 restriction fragment length polymorphism typing.
Abstract: 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.
1,270 citations
TL;DR: The authors have sequenced a total of 109 alleles of the three Y. pestis CRISPRs and they describe 29 new spacers, most being specific to one isolate, and it appears that addition of new motifs to a common ancestral element is the most frequent event.
Abstract: The remarkable repetitive elements called CRISPRs (clustered regularly interspaced short palindromic repeats) consist of repeats interspaced with non-repetitive elements or 'spacers' CRISPRs are present in both archaea and bacteria, in association with genes involved in DNA recombination and repair In the Yersinia pestis genome, three such elements are found at three distinct loci, one of them being highly polymorphic The authors have sequenced a total of 109 alleles of the three Y pestis CRISPRs and they describe 29 new spacers, most being specific to one isolate In nine strains of Yersinia pseudotuberculosis, 132 spacers were found, of which only three are common to Y pestis isolates In Y pestis of the Orientalis biovar investigated in detail here, deletion of motifs is observed but it appears that addition of new motifs to a common ancestral element is the most frequent event This takes place at the three different loci, although at a higher rate in one of the loci, and the addition of new motifs is polarized Interestingly, the most recently acquired spacers were found to have a homologue at another locus in the genome, the majority of these inside an inactive prophage This is believed to be the first time that the origin of the spacers in CRISPR elements has been explained The CRISPR structure provides a new and robust identification tool
1,123 citations
Cites background or methods from "Simultaneous detection and strain d..."
...…relevance of their curious behaviour, analysis of the CRISPRs of Y. pestis provides a tool, comparable to spoligotyping for M. tuberculosis (Kamerbeek et al., 1997), which might open the way to strain typing of ancient, degraded DNA, since a perfectly conserved multi-copy sequence (the…...
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...The high degree of polymorphism generated by the variable spacers forms the basis of the spoligotyping method (Kamerbeek et al., 1997)....
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...tuberculosis (Kamerbeek et al., 1997), which might open the way to strain typing of ancient, degraded DNA, since a perfectly conserved multi-copy sequence (the repeat) can be used to amplify a variable library of very short spacers and since this kind of assay is not sensitive to the occasional misincorporation of an incorrect nucleotide during the amplification process....
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References
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TL;DR: In this article, the authors proposed a standardized technique which exploits variability in both the number and genomic position of IS6110 to generate strain-specific patterns for DNA fingerprinting of Mycobacterium tuberculosis.
Abstract: DNA fingerprinting of Mycobacterium tuberculosis has been shown to be a powerful epidemiologic tool. We propose a standardized technique which exploits variability in both the number and genomic position of IS6110 to generate strain-specific patterns. General use of this technique will permit comparison of results between different laboratories. Such comparisons will facilitate investigations into the international transmission of tuberculosis and may identify specific strains with unique properties such as high infectivity, virulence, or drug resistance.
2,386 citations
TL;DR: The economic costs of not adequately addressing the problem of tuberculosis in this country are estimated from an epidemiological model.
Abstract: Tuberculosis remains the leading cause of death in the world from a single infectious disease, although there is little knowledge of the mechanisms of its pathogenesis and protection from it. After a century of decline in the United States, tuberculosis is increasing, and strains resistant to multiple antibiotics have emerged. This excess of cases is attributable to changes in the social structure in cities, the human immunodeficiency virus epidemic, and a failure in certain major cities to improve public treatment programs. The economic costs of not adequately addressing the problem of tuberculosis in this country are estimated from an epidemiological model.
1,390 citations
TL;DR: Analysis of M. tuberculosis isolates from all patients reported to the tuberculosis registry in San Francisco during 1991 and 1992 confirmed that poorly compliant patients with infectious tuberculosis have a substantial adverse effect on the control of this disease.
Abstract: Background The epidemiology of tuberculosis in urban populations is changing. Combining conventional epidemiologic techniques with DNA fingerprinting of Mycobacterium tuberculosis can improve the understanding of how tuberculosis is transmitted. Methods We used restriction-fragment-length polymorphism (RFLP) analysis to study M. tuberculosis isolates from all patients reported to the tuberculosis registry in San Francisco during 1991 and 1992. These results were interpreted along with clinical, demographic, and epidemiologic data. Patients infected with the same strains were identified according to their RFLP patterns, and patients with identical patterns were grouped in clusters. Risk factors for being in a cluster were analyzed. Results Of 473 patients studied, 191 appeared to have active tuberculosis as a result of recent infection. Tracing of patients' contacts with the use of conventional methods identified links among only 10 percent of these patients. DNA fingerprinting, however, identified 44 clus...
1,116 citations
TL;DR: There should be heightened surveillance for tuberculosis in facilities where HIV-infected persons live, and investigation of contacts must be undertaken promptly and be focused more broadly than is usual.
Abstract: Background. Tuberculosis typically develops from a reactivation of latent infection. Clinical tuberculosis may also arise from a primary infection, and this is thought to be more likely in persons infected with the human immunodeficiency virus (HIV). However, the relative importance of these two pathogenetic mechanisms in this population is unclear. Methods. Between December 1990 and April 1991, tuberculosis was diagnosed in 12 residents of a housing facility for HIV-infected persons. In the preceding six months, two patients being treated for tuberculosis had been admitted to the facility. We investigated this outbreak using standard procedures plus analysis of the cultured organisms with Restriction-Fragment Length polymorphisms (RFLPs). Results. Organisms isolated from all 11 of the culturepositive residents had similar RFLP patterns, whereas the isolates from the 2 patients treated for tuberculosis in the previous six months were different strains. This implicated the first of the 12 patients...
990 citations
"Simultaneous detection and strain d..." refers background in this paper
...Similarly, slight variations in IS6110 fingerprints have occasionally been found among outbreak strains (6, 26, 28)....
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...tuberculosis infection and the accelerated progression of the disease (6), rapid diagnosis and typing of the causative mycobacteria may help to effectively control multidrug-resistant tuberculosis....
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TL;DR: The results indicate that M. tuberculosis strains from regions in central Africa, where tuberculosis is highly prevalent, are generally more related to each other than isolates from the Netherlands, where the transmission rate is low and where the majority of the tuberculosis cases are presumed to be the result of reactivation of previously contracted M.culosis infections.
Abstract: In this study we established the usefulness of DNA fingerprinting for the epidemiology of tuberculosis on the basis of the DNA polymorphism generated by the insertion sequence (IS) IS986. Although clinical isolates of Mycobacterium tuberculosis displayed a remarkably high degree of restriction fragment length polymorphism, we showed that transposition of this IS element is an extremely rare event in M. tuberculosis complex strains grown either in vitro or in vivo for long periods of time. The M. tuberculosis and Mycobacterium africanum strains tested in this study contained 6 to 17 IS copies. In the Mycobacterium bovis strains, the copy numbers ranged between 1 and 5, and all 27 M. bovis BCG strains investigated invariably contained a single IS copy. This copy was located at a unique chromosomal position, reinforcing the idea that the frequency of IS transposition is very low in M. tuberculosis complex strains. Various microepidemics are described in which each microepidemic corresponds to a particular fingerprint type. The extent of similarity between Dutch and African strains was quantitatively assessed by computer-assisted analysis of DNA fingerprints. The results indicate that M. tuberculosis strains from regions in central Africa, where tuberculosis is highly prevalent, are generally more related to each other than isolates from the Netherlands, where the transmission rate is low and where the majority of the tuberculosis cases are presumed to be the result of reactivation of previously contracted M. tuberculosis infections.
989 citations
"Simultaneous detection and strain d..." refers background or methods in this paper
...Similarly, slight variations in IS6110 fingerprints have occasionally been found among outbreak strains (6, 26, 28)....
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...DNA was done by using chemiluminescent ECL (Amersham) detection liquid (28, 29), followed by exposure to X-ray film (Hyperfilm ECL; Amersham) in accordance with the instructions of the manufacturer....
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...purified from cultured cells (28) was used as a target for spoligotyping....
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