Comparison of multilocus variable number tandem repeat analysis and whole genome sequencing for investigation of Clostridium difficile transmission
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REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| ERR347577_1.fastq.gz | Fastqsanger.gz | |||
| ERR347577_2.fastq.gz | Fastqsanger.gz | |||
| ERR347578_1.fastq.gz | Fastqsanger.gz | |||
| ERR347578_2.fastq.gz | Fastqsanger.gz | |||
| ERR347579_1.fastq.gz | Fastqsanger.gz |
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Project description:No study to date has compared multilocus variable-number tandem-repeat analysis (MLVA) and whole-genome sequencing (WGS) in an investigation of the transmission of Clostridium difficile infection. Isolates from 61 adults with ongoing and/or recurrent C. difficile infections and 17 asymptomatic carriage episodes in children (201 samples), as well as from 61 suspected outbreaks affecting 2 to 41 patients in 31 hospitals in the United Kingdom (300 samples), underwent 7-locus MLVA and WGS in parallel. When the first and last samples from the same individual taken for a median (interquartile range [IQR]) of 63 days (43 to 105 days) apart were compared, the estimated rates of the evolution of single nucleotide variants (SNVs), summed tandem-repeat differences (STRDs), and locus variants (LVs) were 0.79 (95% confidence interval [CI], 0.00 to 1.75), 1.63 (95% CI, 0.00 to 3.59), and 1.21 (95% CI, 0.00 to 2.67)/called genome/year, respectively. Differences of >2 SNVs and >10 STRDs have been used to exclude direct case-to-case transmission. With the first serial sample per individual being used to assess discriminatory power, across all pairs of samples sharing a PCR ribotype, 192/283 (68%) differed by >10 STRDs and 217/283 (77%) by >2 SNVs. Among all pairs of cases from the same suspected outbreak, 1,190/1,488 (80%) pairs had concordant results using >2 SNVs and >10 STRDs to exclude transmission. For the discordant pairs, 229 (15%) had ?2 SNVs but ?10 STRDs, and 69 (5%) had ?2 SNVs but ?10 STRDs. Discordant pairs had higher numbers of LVs than concordant pairs, supporting the more diverse measure in each type of discordant pair. Conclusions on whether the potential outbreaks were confirmed were concordant in 58/61 (95%) investigations. Overall findings using MLVA and WGS were very similar despite the fact that they analyzed different parts of the bacterial genome. With improvements in WGS technology, it is likely that MLVA locus data will be available from WGS in the near future.
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Project description:A multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) method was developed and evaluated for the subtyping of Shigella sonnei isolates. A total of 26 VNTR loci were identified by exploring the repeat sequence loci in the genomic sequences of S. sonnei strains Ss046 and 53G and by testing 536 isolates that had previously been characterized by pulsed-field gel electrophoresis (PFGE). The discriminatory power of MLVA (Simpson's index of diversity [D], 0.9524; 95% confidence interval [CI], 0.9373 to 0.9564) for the 536 isolates was significantly higher than that of PFGE (D, 0.8882; CI, 0.8667 to 0.9097). MLVA typing with the four and eight most variable loci had D values of 0.9468 and 0.9481, respectively, results approaching that of 26 loci. The usefulness of MLVA for outbreak investigation was evaluated using 151 isolates from 10 shigellosis outbreaks and 22 PFGE-indistinguishable isolates collected from nine epidemiologically unrelated events in five different countries. The evaluations indicated that MLVA was a powerful typing tool to distinguish isolates for outbreak investigation and that it exhibited a good discrimination of the 22 PFGE-indistinguishable isolates. Single-locus variants did occur during the outbreak; therefore, S. sonnei isolates with MLVA profiles differing at no more than a single locus should be considered part of the same outbreak. The present study suggests that MLVA has the potential to replace PFGE as a standard method of typing S. sonnei isolates for disease surveillance and outbreak investigation.
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Project description:PCR ribotyping is currently used in many countries for epidemiological investigation to track transmission and to identify emerging variants of Clostridium difficile. Although PCR ribotyping differentiates over 300 types, it is not always sufficiently discriminatory for epidemiological investigations particularly for common ribotypes, e.g., ribotypes 027, 106, and 017. Multilocus variable-number tandem-repeat analysis (MLVA) is a highly discriminatory molecular subtyping method that has been applied to a number of bacterial species for high-level subtyping. Two MLVA typing schemes for C. difficile have been previously published, each utilizing seven variable-number tandem-repeat (VNTR) loci on the genome with four loci common to both schemes. Although these schemes are good genotyping methods with the ability to discriminate between isolates, they do not identify the ribotype. We show here that increasing the number of VNTR loci to 15, creating the extended MLVA (eMLVA) scheme, we have successfully subtyped all clinically significant ribotypes while still clustering isolates in concordance with PCR ribotyping. The eMLVA scheme developed here provides insight into the genetic diversity of the C. difficile population at both global and cross-infection clusters in patient levels, with the possibility of replacing PCR ribotyping.
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Project description:The etiology of Crohn's disease in humans is largely unknown. Clinical signs of Crohn's disease partly resemble the clinical picture of Johne's disease in ruminants caused by Mycobacterium avium subsp. paratuberculosis. Because of the high prevalence of these bacteria in (products of) ruminants and their remarkable thermostability, concern has been raised about the possible role of these bacteria in the pathogenesis of Crohn's disease. In an attempt to develop a molecular typing method to facilitate meaningful comparative DNA fingerprinting of M. avium subsp. paratuberculosis isolates from the human and animal reservoirs, multilocus variable-number tandem-repeat analysis (MLVA) was explored and compared to IS900 restriction fragment length polymorphism (RFLP) typing. MLVA typing subdivided the most predominant RFLP type, R01, into six subtypes and thus provides a promising molecular subtyping approach to study the diversity of M. avium subsp. paratuberculosis.
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Project description:BACKGROUND: Shigella flexneri is one of the causative agents of shigellosis, a major cause of childhood mortality in developing countries. Multilocus variable-number tandem repeat (VNTR) analysis (MLVA) is a prominent subtyping method to resolve closely related bacterial isolates for investigation of disease outbreaks and provide information for establishing phylogenetic patterns among isolates. The present study aimed to develop an MLVA method for S. flexneri and the VNTR loci identified were tested on 242 S. flexneri isolates to evaluate their variability in various serotypes. The isolates were also analyzed by pulsed-field gel electrophoresis (PFGE) to compare the discriminatory power and to evaluate the usefulness of MLVA as a tool for phylogenetic analysis of S. flexneri. RESULTS: Thirty-six VNTR loci were identified by exploring the repeat sequence loci in genomic sequences of Shigella species and by testing the loci on nine isolates of different subserotypes. The VNTR loci in different serotype groups differed greatly in their variability. The discriminatory power of an MLVA assay based on four most variable VNTR loci was higher, though not significantly, than PFGE for the total isolates, a panel of 2a isolates, which were relatively diverse, and a panel of 4a/Y isolates, which were closely-related. Phylogenetic groupings based on PFGE patterns and MLVA profiles were considerably concordant. The genetic relationships among the isolates were correlated with serotypes. The phylogenetic trees constructed using PFGE patterns and MLVA profiles presented two distinct clusters for the isolates of serotype 3 and one distinct cluster for each of the serotype groups, 1a/1b/NT, 2a/2b/X/NT, 4a/Y, and 6. Isolates that had different serotypes but had closer genetic relatedness than those with the same serotype were observed between serotype Y and subserotype 4a, serotype X and subserotype 2b, subserotype 1a and 1b, and subserotype 3a and 3b. CONCLUSIONS: The 36 VNTR loci identified exhibited considerably different degrees of variability among S. flexneri serotype groups. VNTR locus could be highly variable in a serotype but invariable in others. MLVA assay based on four highly variable loci could display a comparable resolving power to PFGE in discriminating isolates. MLVA is also a prominent molecular tool for phylogenetic analysis of S. flexneri; the resulting data are beneficial to establish clear clonal patterns among different serotype groups and to discern clonal groups among isolates within the same serotype. As highly variable VNTR loci could be serotype-specific, a common MLVA protocol that consists of only a small set of loci, for example four to eight loci, and that provides high resolving power to all S. flexneri serotypes may not be obtainable.
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