Sequetyping: Serotyping Streptococcus pneumoniae by a Single PCR Sequencing Strategy

Leung, Marcus H. Y.; Bryson, Kevin; Freystatter, Kathrin; Pichon, Bruno; Edwards, Giles; Charalambous, Bambos M.; Gillespie, Stephen H.

doi:10.1128/jcm.06384-11

Cited by 64 publications

(74 citation statements)

References 68 publications

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“…A confirmatory test with a serotype-specific PCR targeting wzy has been recently introduced because of the possibility that serotype switching could dissociate previously identified cpsA-cpsB sequence types from serotypes identified by wzy PCR, especially in a highly immunized population (257). Another group used a similar approach based on a region of cpsB (258).…”

Section: Genotyping Approachesmentioning

confidence: 99%

Pneumococcal Capsules and Their Types: Past, Present, and Future

et al. 2015

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SUMMARY Streptococcus pneumoniae (the pneumococcus) is an important human pathogen. Its virulence is largely due to its polysaccharide capsule, which shields it from the host immune system, and because of this, the capsule has been extensively studied. Studies of the capsule led to the identification of DNA as the genetic material, identification of many different capsular serotypes, and identification of the serotype-specific nature of protection by adaptive immunity. Recent studies have led to the determination of capsular polysaccharide structures for many serotypes using advanced analytical technologies, complete elucidation of genetic basis for the capsular types, and the development of highly effective pneumococcal conjugate vaccines. Conjugate vaccine use has altered the serotype distribution by either serotype replacement or switching, and this has increased the need to serotype pneumococci. Due to great advances in molecular technologies and our understanding of the pneumococcal genome, molecular approaches have become powerful tools to predict pneumococcal serotypes. In addition, more-precise and -efficient serotyping methods that directly detect polysaccharide structures are emerging. These improvements in our capabilities will greatly enhance future investigations of pneumococcal epidemiology and diseases and the biology of colonization and innate immunity to pneumococcal capsules.

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Section: Genotyping Approachesmentioning

confidence: 99%

Pneumococcal Capsules and Their Types: Past, Present, and Future

et al. 2015

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“…[19,[34][35][36][37][38][39] For example, while serotype 7F is covered in all three pneumococcal vaccines, 7F cannot be discriminated from serotype 7A using these molecular methods, and therefore the serotype is assigned 7F/7A. Other vaccinepreventable serotypes identified by PCR-based serotyping cannot discriminate: 6A from 6B; 9V from 9A; 9N from 9L; 11A from 11D; 12F from serotypes 12A, 12B, 44, and 46; 15B from 15C; 18C from serotypes 18F, 18B, and 18A; 22F from 22A; 33F from serotypes 33A and 37.…”

Section: Discussionmentioning

confidence: 99%

Refining PCR-based serotyping for detection of vaccine-preventable Streptococcus pneumoniae

Lang

Gillis

ElSherif

et al. 2016

JER

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Conventional multiplex PCR (cmPCR) reactions have been developed to monitor the most predominant serotypes of Streptococcus pneumoniae causing invasive pneumococcal disease (IPD). Since cmPCR assigns serotypes based on differences in the capsule biosynthesis (cps) loci, DNA extracted from clinical specimens can be used directly to monitor changes in serotype distribution and assess the impact of pneumococcal vaccines. Given that cmPCR can require up to eight reactions to assign a serotype, testing is often conducted in sequential algorithms. Sequential cmPCR reactions; however, may not be the most cost effective strategy to determine whether a S. pneumoniae serotype is vaccine-preventable. This study used oligonucleotide permutations in a modified set of cmPCR reactions (termed cmPCRmod) to reduce the number of PCR reactions required to identify S. pneumoniae serotypes covered by the 7-and 13-valent pneumococcal conjugate vaccines (PCV7 and PCV13, respectively) and the 23-valent pneumococcal polysaccharide vaccine (PPV23). While oligonucleotide permutations have previously been reported for regional differences in serotype distribution, the impact on assay performance had not been assessed. This study demonstrated that equivalent analytical sensitivity and specificity was seen when comparing cmPCR and cmPCRmod, and 100% concordance was seen when 308 clinical isolates of S. pneumoniae were evaluated. Compared to cmPCR, cmPCRmod reduced the number and reactions required to detect serotypes covered by PCV7, PCV13, and PPV23. This study demonstrated that conventional multiplex reactions can be reformulated for more efficient detection of vaccine-preventable serotypes, without compromising test performance characteristics. As such, cmPCRmod reactions could provide significant cost savings for large surveillance studies.

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“…In general, PCR-based typing assays, such as those designed for Streptococcus pneumoniae (13), Neisseria meningitidis (14), and Acinetobacter baumannii (15), are desirable because of their inherent advantages of low cost and high speed. The expense comprises the cost of standard PCR reagents, and the turnaround time consists of only the few hours needed to prepare DNA and run the PCR.…”

Section: Discussionmentioning

confidence: 99%

“…Several groups are attempting to take advantage of existing genome sequence data to design sequence-and PCR-based typing assays. In the last year, PCR-based typing assays have been published for organisms such as Streptococcus pneumoniae (13), Neisseria meningitidis (14), and Acinetobacter baumannii (15).…”

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confidence: 99%

Pan-PCR, a Computational Method for Designing Bacterium-Typing Assays Based on Whole-Genome Sequence Data

et al. 2013

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cWith increasing rates of antibiotic resistance, bacterial infections have become more difficult to treat, elevating the importance of surveillance and prevention. Effective surveillance relies on the availability of rapid, cost-effective, and informative typing methods to monitor bacterial isolates. PCR-based typing assays are fast and inexpensive, but their utility is limited by the lack of targets which are capable of distinguishing between strains within a species. To identify highly informative PCR targets from the growing base of publicly available bacterial genome sequences, we developed pan-PCR. This computer algorithm uses existing genome sequences for isolates of a species of interest and identifies a set of genes whose patterns of presence or absence provide the best discrimination between strains in this species. A set of PCR primers targeting the identified genes is then designed, with each PCR product being of a different size to allow multiplexing. These target DNA regions and PCR primers can then be utilized to type bacterial isolates. To evaluate pan-PCR, we designed an assay for the emerging pathogen Acinetobacter baumannii. Taking as input a set of 29 previously sequenced genomes, pan-PCR identified 6 genetic loci whose presence or absence was capable of distinguishing all the input strains. This assay was applied to a set of patient isolates, and its discriminatory power was compared to that of multilocus sequence typing (MLST) and whole-genome optical maps. We found that the pan-PCR assay was capable of making clinically relevant distinctions between strains with identical MLST profiles and showed a discriminatory power similar to that of optical maps. Pan-PCR represents a tool capable of exploiting available genome sequence data to design highly discriminatory PCR assays. The ease of design and implementation makes this approach feasible for diagnostic facilities of all sizes.

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Sequetyping: Serotyping Streptococcus pneumoniae by a Single PCR Sequencing Strategy

Cited by 64 publications

References 68 publications

Pneumococcal Capsules and Their Types: Past, Present, and Future

Pneumococcal Capsules and Their Types: Past, Present, and Future

Refining PCR-based serotyping for detection of vaccine-preventable Streptococcus pneumoniae

Pan-PCR, a Computational Method for Designing Bacterium-Typing Assays Based on Whole-Genome Sequence Data

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