PCR-based discrimination of emerging Streptococcus pneumoniae serotypes 22F and 33F

Gillis, Hayley D; Demczuk, Walter; Griffith, Averil; Martín, Irene; Warhuus, Michelle; Lang, Amanda; ElSherif, May; McNeil, Shelly; LeBlanc, Jason J.

doi:10.1016/j.mimet.2017.11.017

Cited by 9 publications

(3 citation statements)

References 52 publications

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“…The novel DPO, LNA, and other assay-sets designed here, combined with previously published serogroup 6, 18, and 22 assay-sets effectively discriminated between current PCV-formulation (PCV10, PCV13) VT (6A and 6B, 18C), next-generation PCV-formulation (PCV15, PCV20) VT 22F and NVT (6C, 6D, 18A, 18B, 18F and 22A) within a nanofluidic molecular serotyping reaction-set. Serogroups 6, 18, and 22 have previously been discriminated using conventional PCR 15 , 27 , 28 , or sequencing-based methods 12 , 13 , 29 . Sequencing-based methods are expensive, not all laboratories have access to sequencing platforms, bioinformatics software and expertise.…”

Section: Discussionmentioning

confidence: 99%

High-throughput nanofluidic real-time PCR to discriminate Pneumococcal Conjugate Vaccine (PCV)-associated serogroups 6, 18, and 22 to serotypes using modified oligonucleotides

Downs

Madhi

Merwe

et al. 2021

Sci Rep

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Current real-time high-throughput Polymerase Chain Reaction (qPCR) methods do not distinguish serotypes 6A from 6B, 18C from 18A/B and 22F from 22A. We established a nanofluidic real-time PCR (Fluidigm) for serotyping that included Dual-Priming-Oligonucleotides (DPO), a Locked-Nucleic-Acid (LNA) probe and TaqMan assay-sets for high-throughput serotyping. The designed assay-sets target capsular gene wciP in serogroup 6, wciX and wxcM in serogroup 18, and wcwA in serogroup 22. An algorithm combining results from published assay-sets (6A/B/C/D; 6C/D; 18A/B/C; 22A/F) and designed assay-sets for 6A/C; 18B/C/F; 18C/F, 18F and 22F was validated through blind analysis of 1973 archived clinical samples collected from South African children ≤ 5-years-old (2009–2011), previously serotyped with the culture-based Quellung method. All assay-sets were efficient (92–101%), had low variation between replicates (R2 > 0.98), and were able to detect targets at a limit of detection (LOD) of < 100 Colony-Forming-Units (CFU)/mL of sample. There was high concordance (Kappa = 0.73–0.92); sensitivity (85–100%) and specificity (96–100%) for Fluidigm compared with Quellung for serotyping 6A; 6B; 6C; 18C and 22F. Fluidigm distinguishes vaccine-serotypes 6A, 6B, 18C, next-generation PCV-serotype 22F and non-vaccine-serotypes 6C, 6D, 18A, 18B, 18F and 22A. Discriminating single serotypes is important for assessing serotype replacement and the impact of PCVs on vaccine- and non-vaccine serotypes.

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Section: Discussionmentioning

confidence: 99%

High-throughput nanofluidic real-time PCR to discriminate Pneumococcal Conjugate Vaccine (PCV)-associated serogroups 6, 18, and 22 to serotypes using modified oligonucleotides

Downs

Madhi

Merwe

et al. 2021

Sci Rep

View full text Add to dashboard Cite

show abstract

“…The novel DPO, LNA, and other assay-sets designed here, combined with previously published serogroup 6, 18, and 22 assay-sets effectively discriminated between current PCV-formulation (PCV10, PCV13) VT (6A and 6B, 18C), next-generation PCV-formulation (PCV15, PCV20) VT 22F and NVT (6C, 6D, 18A, 18B, 18F and 22A) within a nano uidic molecular serotyping reaction-set. Serogroups 6, 18, and 22 have previously been discriminated using conventional PCR 15,26,27 , or sequencing-based methods 12,13,28 . Sequencing-based methods are expensive, not all laboratories have access to sequencing platforms, bioinformatics software and expertise.…”

Section: Discussionmentioning

confidence: 99%

High-throughput nanofluidic real-time PCR to discriminate Pneumococcal Conjugate Vaccine (PCV)-associated serogroups 6, 18, and 22 to serotypes using modified oligonucleotides.

Downs

Madhi

Merwe

et al. 2021

Preprint

View full text Add to dashboard Cite

Current real-time Polymerase Chain Reaction (qPCR) methods are unable to distinguish serotypes 6A from 6B, 18C from 18A/B and 22F from 22A. We established a nanofluidic real-time PCR (Fluidigm) for serotyping that included Dual-Priming-Oligonucleotides (DPO), a Locked-Nucleic-Acid (LNA) probe and TaqMan assay-sets for high-throughput serotyping. The designed assay-sets target capsular gene wciP in serogroup 6, wciX and wxcM in serogroup 18, and wcwA in serogroup 22. An algorithm combining results from published assay-sets (6A/B/C/D; 6C/D; 18A/B/C; 22A/F) and designed assay-sets for 6A/C; 18B/C/F; 18C/F, 18F and 22F was validated through blind analysis of 1973 archived clinical samples collected from South African children ≤ 5-years-old (2009-11), previously serotyped with the culture-based Quellung method. All assay-sets were efficient (92–101%), had low variation between replicates (R2 > 0.98), and were able to detect targets at a limit of detection (LOD) of < 100 Colony-Forming-Units (CFU)/ml of sample. There was high concordance (Kappa = 0.73–0.92); sensitivity (85–100%) and specificity (96–100%) for Fluidigm compared with Quellung for serotyping 6A; 6B; 6C; 18C and 22F. Fluidigm distinguishes vaccine-serotypes 6A, 6B, 18C, next-generation PCV-serotype 22F and non-vaccine-serotypes 6C, 6D, 18A, 18B, 18F and 22A. Discriminating single serotypes is important for assessing serotype replacement and the impact of PCVs on vaccine- and non-vaccine serotypes.

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“…Isolates were typed using the modified scheme of sequential multiplex PCR protocol with sequential reactions described previously [30][31][32]. The serotypes studied were: 19A, 19F, 20, 22F/A, 23A, 23B, 23F, 31, 33F, 34, 35B, 35F/47F, 37 and 38/25F.…”

Section: Capsular Typingmentioning

confidence: 99%

Prevalence and clinical impact of Streptococcus pneumoniae nasopharyngeal carriage in solid organ transplant recipients

Roca-Oporto

Cebrero-Cangueiro

Gil-Marqués

et al. 2019

Preprint

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Background: S. pneumoniae is the leading cause of community-acquired pneumonia in the solid organ transplant recipient (SOTR); nevertheless, the prevalence of colonization and of the colonizing/infecting serotypes has not been studied in this population. In this context, the aim of the present study was to describe the rate, characteristics, and clinical impact of S. pneumoniae nasopharyngeal carriage. Methods: A prospective observational cohort of Solid Organ Transplant recipients (SOTR) was held at the University Hospital Virgen del Rocío, Seville, Spain with the aim to evaluate the S. pneumoniae colonization and the serotype prevalence in SOTR. Two different pharyngeal swabs samples from 500 patients were included in two different seasonal periods winter and spring/summer. Optochin and bile solubility tests were performed for the isolation of thew strains. Antimicrobial susceptibility studies (MICs, mg/l) of levofloxacin, trimethoprim-sulfamethoxazole, penicillin, amoxicillin, cefotaxime, ceftriaxone, erythromycin, azithromycin and vancomycin for each isolate were determined by E-test strips. Capsular typing was done by sequential multiplex PCR reactions. A multivariate logistic regression analysis of factors potentially associated with pneumococcal nasopharyngeal carriage and disease was performed. Results: Twenty-six (5.6%) and fifteen (3.2%) patients were colonized in winter and spring/summer periods, respectively. Colonized SOT recipients compared to non-colonized patients were more frequently men (79.5% vs. 63.1%, P<0.05) and cohabitated regularly with children (59% vs. 32.2%, P<0.001). The most prevalent serotype in both studied periods was 35B. Forty-five percent of total isolates were included in the pneumococcal vaccine PPV23. Trimethoprim-sulfamethoxazole and macrolides were the less active antibiotics. Three patients had non-bacteremic pneumococcal pneumonia, and two of them died. Conclusions: Pneumococcal colonization in SOTR is low with the most colonizing serotypes not included in the pneumococcal vaccines.

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PCR-based discrimination of emerging Streptococcus pneumoniae serotypes 22F and 33F

Cited by 9 publications

References 52 publications

High-throughput nanofluidic real-time PCR to discriminate Pneumococcal Conjugate Vaccine (PCV)-associated serogroups 6, 18, and 22 to serotypes using modified oligonucleotides

High-throughput nanofluidic real-time PCR to discriminate Pneumococcal Conjugate Vaccine (PCV)-associated serogroups 6, 18, and 22 to serotypes using modified oligonucleotides

High-throughput nanofluidic real-time PCR to discriminate Pneumococcal Conjugate Vaccine (PCV)-associated serogroups 6, 18, and 22 to serotypes using modified oligonucleotides.

Prevalence and clinical impact of Streptococcus pneumoniae nasopharyngeal carriage in solid organ transplant recipients

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