Concurrent Serotyping and Genotyping of Pneumococci by Use of PCR and Electrospray Ionization Mass Spectrometry

Massire, Christian; Gertz, Robert E.; Svoboda, Pavel; Levert, Keith; Reed, Matthew S.; Pohl, Jan; Kreft, Rachel; Feng, Li; White, Neill; Ranken, Ray; Blyn, Larry B.; Ecker, David J.; Sampath, Rangarajan; Beall, Bernard

doi:10.1128/jcm.06735-11

Cited by 23 publications

(12 citation statements)

References 23 publications

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“…Amplicon base composition is analyzed by electrospray ionization MS to predict 45 serotypes/serogroups and identify the MLST with good reliability (230). A similar principle was used in a MassTag PCR (231), in which primers were conjugated with individual low-molecular-weight tags so that amplicons with 2 corresponding tags were identified by MS. Five mPCRs, each with 8 to 12 primer pairs (mainly targeting wzy), can identify all serotypes/serogroups, including 28 individually.…”

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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“…These methods include a multiplexed PCR coupled to an automated microarray assay differentiating 22 serotypes and 24 other serotypes to the subgroup level, the sequetyping method, which relies on sequencing products of a single consensus pair of primers capable of amplifying products of 84 serotypes and differentiating 46, a 5-plex multiplex PCR followed by ionization mass spectrometry, which is capable of differentiating 45 serotypes, and a set of three multiplex PCRs with 40 pairs of previously described primers followed by fragment analysis using automated fluorescence-based capillary electrophoresis, which is capable of differentiating 39 serotype/serogroups (15)(16)(17). These methods are similar to our current method in terms of serotype resolution; however, to the best of our knowledge this is the first instance in which NGS coupled with target enrichment has been used to determine S. pneumoniae with serogroups directly from clinical specimens, although recently a similar method was used to identify other bacterial isolates (18).…”

Section: Discussionmentioning

confidence: 99%

Direct Detection and Prediction of All Pneumococcal Serogroups by Target Enrichment-Based Next-Generation Sequencing

Liyanapathirana

Ang

et al. 2014

J Clin Microbiol

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Despite the availability of standard methods for pneumococcal serotyping, there is room for improvement in the available methods, in terms of throughput, multiplexing capacity, and the number of serotypes identified. We describe a target enrichmentbased next-generation sequencing method applied to nasopharyngeal samples for direct detection and serogroup prediction of all known serotypes of Streptococcus pneumoniae, 32 to the serotype level and the rest to the closely related serogroup level. The method was applied to detect and to predict the serogroups of pneumococci directly in clinical samples and from sweeps of primary culture DNA, with increased detection rates versus culture-based identification and agreement with the serotypes/serogroups determined by conventional serotyping methods. We propose this method, in conjunction with traditional serotyping methods, as an alternative to rapid detection and serotyping of pneumococci. The introduction of conjugate vaccination has dramatically altered the prevalence and community structure of pneumococcal serotypes, in both disease and carriage (1). As the serotype valency of conjugate vaccines increased from 7 to 13, the serotypes and their prevalence were subjected to dynamic changes to less common serotypes. Thus, there is a need for laboratory methods that are capable of identifying the maximum possible number of serotypes with a limited number of assays, in order to monitor serotype replacement and any emerging serotypes (2).The Quellung reaction remains the standard method for identification of pneumococcal serotypes. This method is expensive and time-consuming and requires expertise (3). With the sequencing of the capsular biosynthesis loci of all 90 pneumococcal serotypes, new molecular methods for pneumococcal serotyping have been developed (4). The most widely used of these methods remain sequential multiplex PCRs. The Centers for Disease Control and Prevention (CDC) (Atlanta, GA) recommends a set of 8 multiplex PCRs that are capable of differentiating 40 seroidentities, 22 to the serotype level (5) (http://www.cdc.gov/streplab/pcr .html). More recently, a set of seven real-time PCR assays capable of differentiating 21 serotypes was also recommended by the CDC (6). However, the number of tests to be performed still remains relatively high, due to the limited multiplexing capabilities associated with gel-based differentiation and quencher dye combinations. Thus, there is a need for alternative serotyping methods capable of differentiating the greatest number of serotypes at least to closely related serogroups, with a minimal number of assays, in a rapid and cost-effective manner.Next-generation sequencing (NGS) is an attractive alternative platform for the development of diagnostic methods. The high throughput, the increasingly simple and fast methods for sample preparation, and the ability to pool samples together make these platforms versatile for adaptation. Target enrichment-based sequencing through selective enrichment of the regions of interest enabl...

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“…Other limitations of the use of PCR/ESI-TOF-MS in this context include the possibility of detection of nonviable organisms, cost, the semi-quantitative nature of the data, and inability to recover specific strains linked to a patient or outbreak isolate. In this study, PCR/ESI-TOF-MS was not used to detect specific strains of bacteria detected, though it can be and has been used specifically for rapid genotyping of A. baumannii [21], S. aureus [22] and Streptococcus pneumoniae [38]. Thus, this technology may yet prove useful in outbreak investigations using environmental sampling, especially since it detected 4–5 fold higher numbers of pathogens per site without adversely affecting the ratio of clinically irrelevant microbes.…”

Section: Discussionmentioning

confidence: 99%

Comparison of PCR/Electron spray Ionization-Time-of-Flight-Mass Spectrometry versus Traditional Clinical Microbiology for active surveillance of organisms contaminating high-use surfaces in a burn intensive care unit, an orthopedic ward and healthcare workers

et al. 2012

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BackgroundUnderstanding nosocomial pathogen transmission is restricted by culture limitations. Novel platforms, such as PCR-based electron spray ionization-time-of-flight-mass spectrometry (ESI-TOF-MS), may be useful as investigational tools.MethodsTraditional clinical microbiology (TCM) and PCR/ESI-TOF-MS were used to recover and detect microorganisms from the hands and personal protective equipment of 10 burn intensive care unit (ICU) healthcare workers providing clinical care at a tertiary care military referral hospital. High-use environmental surfaces were assessed in 9 burn ICU and 10 orthopedic patient rooms. Clinical cultures during the study period were reviewed for pathogen comparison with investigational molecular diagnostic methods.ResultsFrom 158 samples, 142 organisms were identified by TCM and 718 by PCR/ESI-TOF-MS. The molecular diagnostic method detected more organisms (4.5 ± 2.1 vs. 0.9 ± 0.8, p < 0.01) from 99% vs. 67% of samples (p < 0.01). TCM detected S. aureus in 13 samples vs. 21 by PCR/ESI-TOF-MS. Gram-negative organisms were less commonly identified than gram-positive by both methods; especially by TCM. Among all detected bacterial species, similar percentages were typical nosocomial pathogens (18-19%) for TCM vs. PCR/ESI-TOF-MS. PCR/ESI-TOF-MS also detected mecA in 112 samples, vanA in 13, and KPC-3 in 2. MecA was associated (p < 0.01) with codetection of coagulase negative staphylococci but not S. aureus. No vanA was codetected with enterococci; one KPC-3 was detected without Klebsiella spp.ConclusionsIn this pilot study, PCR/ESI-TOF-MS detected more organisms, especially gram-negatives, compared to TCM, but the current assay format is limited by the number of antibiotic resistance determinants it covers. Further large-scale assessments of PCR/ESI-TOF-MS for hospital surveillance are warranted.

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Concurrent Serotyping and Genotyping of Pneumococci by Use of PCR and Electrospray Ionization Mass Spectrometry

Cited by 23 publications

References 23 publications

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

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

Direct Detection and Prediction of All Pneumococcal Serogroups by Target Enrichment-Based Next-Generation Sequencing

Comparison of PCR/Electron spray Ionization-Time-of-Flight-Mass Spectrometry versus Traditional Clinical Microbiology for active surveillance of organisms contaminating high-use surfaces in a burn intensive care unit, an orthopedic ward and healthcare workers

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