Serotyping of Streptococcus pneumoniae Isolates from Nasopharyngeal Samples: Use of an Algorithm Combining Microbiologic, Serologic, and Sequential Multiplex PCR Techniques

Miernyk, Karen; DeByle, Carolynn; Harker-Jones, Marcella; Hummel, K; Hennessy, Thomas W.; Wenger, Jay D.; Rudolph, Karen

doi:10.1128/jcm.00610-11

Cited by 26 publications

(17 citation statements)

References 22 publications

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“…Our assay includes serotype 2, which, although rare among U.S. disease isolates, is a significant cause of meningitis in Bangladesh (21) and Mongolia (our unpublished data). Another useful rmPCR assay (22) (7) 19F (7) 14 (6) 14 (6) (13) 4, 19A (13 each) 12F/12A/12B/44/46 (9) 12F/12A/12B/44/46 (9) 6A/6B (8) 6A/6B (8) 14 (6) 14 ( (14) a Serotypes detected by cmPCR but not present in the real-time triplex reactions are underlined. b These cmPCR and rmPCR reactions were all performed employing African schemes (described for the cmPCR assay at http://www.cdc.gov/ncidod/biotech/files/pcr-Africaclinical-specimens.pdf).…”

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confidence: 99%

Sequential Triplex Real-Time PCR Assay for Detecting 21 Pneumococcal Capsular Serotypes That Account for a High Global Disease Burden

et al. 2013

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cWe developed and validated a real-time PCR assay consisting of 7 triplexed reactions to identify 11 individual serotypes plus 10 small serogroups representing the majority of disease-causing isolates of Streptococcus pneumoniae. This assay targets the 13 serotypes included within the 13-valent conjugate vaccine and 8 additional key serotypes or serogroups. Advantages over other serotyping assays are described. The assay will be expanded to 40 serotypes/serogroups. We will provide periodic updates at our protocol website.

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confidence: 99%

Sequential Triplex Real-Time PCR Assay for Detecting 21 Pneumococcal Capsular Serotypes That Account for a High Global Disease Burden

et al. 2013

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“…previously observed [12] did not appear to raise a problem in this setting. The 2 invasive and 20 carrier isolates, undetectable with primers targeting the 19F wzy gene, were further characterized with a primer set targeting the 19A mnaA gene, used previously in the original typing scheme by Pai et al [7] for the detection of serotype 19A.…”

Section: Resultsmentioning

confidence: 99%

PCR deduction of invasive and colonizing pneumococcal serotypes from Venezuela: a critical appraisal

González

Rivera-Olivero

Sisco

et al. 2014

J Infect Dev Ctries

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Introduction: Serotype surveillance of Streptococcus pneumoniae is indispensable for evaluating the potential impact of pneumococcal conjugate vaccines. Serotyping by the standard Quellung reaction is technically demanding, time consuming, and expensive. A simple and economical strategy is multiplex PCR-based serotyping. We evaluated the cost effectiveness of a modified serial multiplex PCR (mPCR), resolving 24 serotypes in four PCR reactions and optimally targeting the most prevalent invasive and colonizing pneumococcal serotypes found in Venezuela. Methodology: A total of 223 pneumococcal isolates, 140 invasive and 83 carriage isolates, previously serotyped by the Quellung reaction and representing the 18 most common serotypes/groups identified in Venezuela, were serotyped with the adapted mPCR. Results: The mPCR serotyped 76% of all the strains in the first two PCR reactions and 91% after four reactions, correctly identifying 17 serotypes/groups. An isolate could be serotyped with mPCR in less than 2 minutes versus 15 minutes for the Quellung reaction, considerably lowering labor costs. A restrictive weakness of mPCR was found for the detection of 19F strains. Most Venezuelan 19F strains were not typeable using the mPCR, and two 19F cps serotype variants were identified. Conclusions: The mPCR assay is an accurate, rapid, and economical method for the identification of the vast majority of the serotypes from Venezuela and can be used in place of the standard Quellung reaction. An exception is the identification of serotype 19F. In this setting, most 19F strains were not detectable with mPCR, demonstrating a need of serology-based quality control for PCR-based serotyping.

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“…The CDC serotyping schemes have been modified in different ways for use not only in Finland but in several other countries as well, which indicates that the oligonucleotide primers used for amplification can be combined in several ways (10,15,23,24). This flexibility is important, as it allows the modification of a scheme according to the serotype distribution of the material to be serotyped, for example, that originating from different geographical regions of the world or representing colonizing or invasive isolates.…”

Section: Discussionmentioning

confidence: 99%

“…This flexibility is important, as it allows the modification of a scheme according to the serotype distribution of the material to be serotyped, for example, that originating from different geographical regions of the world or representing colonizing or invasive isolates. Optimization should also take into account that the amplicons combined in any given mPCR should be of sufficiently different sizes to facilitate visual interpretation, an issue outlined by Miernyk et al (23). For accurate serotyping by PCR, complementary phenotypic methods such as Quellung testing are needed for the subtyping and confirmation of the serotypes that are not distinguishable due to primer cross-reactivity.…”

Section: Discussionmentioning

confidence: 99%

From Quellung to Multiplex PCR, and Back When Needed, in Pneumococcal Serotyping

et al. 2012

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All currently available vaccines against Streptococcus pneumoniae are based on selections of the over 90 different serotypes, which underlines the importance of serotyping for surveillance and vaccine efficacy monitoring. In this study, we modified and validated a PCR-based scheme for deducing the serotypes of the invasive pneumococci isolated in Finland. For validation, 170 isolates were serotyped using the new protocol with six sequential multiplex PCRs for the deduction of serotypes, supplemented with Quellung testing when needed. The results were compared with those obtained by traditional serotyping methods. We found that 98.8% (168/170) of the isolates were correctly serotyped by the new protocol. Subsequently, the scheme was taken into regular use for serotyping the invasive pneumococci isolated in Finland for serotype-specific surveillance purposes and has been applied in the serotyping of more than 1,500 invasive isolates so far. The sequential multiplex PCRs (mPCRs) have given a result for over 99% of the isolates and allowed us to both handle samples in bulk and noticeably reduce the cost of reagents. While serotyping primarily by PCR is precise and effective, Quellung testing remains the most reliable way to discover possible discrepancies between the DNA deduced and the phenotypic serotype of an isolate. Since implementing the protocol for regular use, two serotype 19F PCR-positive isolates were found to be serotype 19A by the Quellung reaction. While a rare occurrence, this is an important observation, which prompted a revision of our serotyping protocol to prevent possible underreporting of serotype 19A, a potential replacement serotype following large-scale vaccination.

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Serotyping of Streptococcus pneumoniae Isolates from Nasopharyngeal Samples: Use of an Algorithm Combining Microbiologic, Serologic, and Sequential Multiplex PCR Techniques

Cited by 26 publications

References 22 publications

Sequential Triplex Real-Time PCR Assay for Detecting 21 Pneumococcal Capsular Serotypes That Account for a High Global Disease Burden

Sequential Triplex Real-Time PCR Assay for Detecting 21 Pneumococcal Capsular Serotypes That Account for a High Global Disease Burden

PCR deduction of invasive and colonizing pneumococcal serotypes from Venezuela: a critical appraisal

From Quellung to Multiplex PCR, and Back When Needed, in Pneumococcal Serotyping

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