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Category: Bacterial Pathogenesis; Viruses and Viral Pathogenesis
The Evolution of Genotyping Strategies to Detect, Analyze, and Control Transmission of Tuberculosis, Page 1 of 2
< Previous page | Next page > /docserver/preview/fulltext/10.1128/9781555819743/9781555819736_Chap13-1.gif /docserver/preview/fulltext/10.1128/9781555819743/9781555819736_Chap13-2.gifAbstract:
The introduction of DNA fingerprinting tools for the analysis of Mycobacterium tuberculosis (MTB) isolates has transformed the way we control the transmission of this pathogen. As with any other infection transmitted via aerosolized particles harboring infective bacteria, obtaining accurate data on the index case and subsequent contacts involved in a transmission chain is challenging.
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Schematic representation of the chromosome of a hypothetical M. tuberculosis complex isolate with marked repetitive elements as targets for different typing methods. The principle of those methods is pictorially outlined. (1) In IS6110-RFLP typing, mycobacterial DNA is cleaved with the restriction endonuclease PvuII, and the resulting fragments are separated electrophoretically on an agarose gel, transferred onto a nylon membrane by Southern blotting, and hybridized to a probe complementary to the 3′ end of the IS6110 (probe target), yielding a characteristic banding pattern in which every band represents a single IS6110 element. (2) Spoligotyping relies upon PCR amplification of a single DR locus that harbors 36-bp DRs interspersed with unique 34- to 41-bp spacer sequences. The PCR products (red horizontal lines) are hybridized to a membrane containing 43 oligonucleotides corresponding to the spacers from M. tuberculosis H37Rv and Mycobacterium bovis BCG. The presence or absence of each of those 43 spacers in the DR region of the analyzed isolate will be represented as the pattern of positive or negative hybridization signals. (3) The VNTR loci or MIRUs are PCR-amplified, and the obtained products (yellow horizontal line) are sized on agarose gels to deduce the number of repeats in each individual locus. (4, 5) Two PCR-based typing methods, that is, DRE-PCR and amplityping, are designed to amplify DNA between clusters of IS6110 and polymorphic GC-rich sequences (PGRS) or between clusters of IS6110 elements, respectively. Different distances between the repetitive elements and their different copy numbers result in variability of banding patterns, composed of DNA fragments amplified (a to d) and produced for individual isolates. Other typing methods (less frequently used) are also shown: heminested inverse PCR (6) and ligation-mediated PCR (7). Figure reprinted and legend adapted from reference 83 , CC BY 3.0.
Schematic representation of how the chronologies of transmission can be inferred from the analysis of SNPs from clustered isolates. Each dot represents a SNP. Each box represents a patient. (A) Hypothetical transmission involving five patients, each differing in one SNP with the closest isolate. Neither the directionality of the transmission nor the index case can be inferred. (B) Hypothetical transmission involving seven patients. If the epidemiological data allow us to determine the index case, the direction of transmission (indicated by arrows) can be deduced.
Different networks of clusters based on the analysis of SNPs obtained from WGS. Each black dot represents a SNP. All the isolates sharing identical SNP composition are included in the same circle. The size of the circle is proportional to the number of isolates included. An example of star-like topology, expected for networks including superspreader case, can be found in the cluster with the central node highlighted in red (second line, leftside). Reprinted from reference 63 with permission from Elsevier CC-BY-4.0.
Chart illustrating work flow between the clinical setting, the genome and analysis center, and the TB research laboratory. (1) Strain X is identified as belonging to an uncontrolled transmission cluster. Strains are analyzed from raw sequencing data (2) to detect polymorphisms at the genome sequencing center. (3) SNPs are detected after comparison of the strain X sequence with those of the reference strains. SNP1 is shared by the strains belonging to the cluster and is not present in the global strain collection. (4) The TB research laboratory will use the transmission cluster-specific SNP to design specific assays. An allele-specific-oligonucleotide PCR assay, TRAP, was chosen to distinguish between strains belonging to the cluster. Once the assay is validated, it is easily transferred to a local clinical setting (5) for screening of ongoing surveillance (both from culture and from direct samples) of the spread of the targeted highly transmissible strains and as support to the local TB control program. Reprinted from reference 68 with permission.