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Microbial Genomics logo Microbial Genomics

Volume 11, Issue 6

Utilizing large and diverse bacterial genome datasets to improve the detection and identification of via PCR-based diagnostics Open Access

Abstract

The accurate identification of (pneumococcus) is crucial for diagnostics and surveillance but is complicated by the use of molecular assays that may also detect non-pneumococcal (NPS) species. Therefore, the aim of this study was to use a combination of and analyses to evaluate PCR assays for the molecular detection and identification of pneumococci. A diverse dataset of over 9,300 pneumococcal and NPS genomes was investigated to determine the sensitivity and specificity of assays for seven recommended gene targets: , , , , Spn9802, SP2020 and Xisco. These findings were used to design new diagnostic assays for two targets, Xisco and SP2020. The new assays were evaluated using three sets of isolates, one of which was selected based upon evidence for sequence diversity from a second investigation of over 6,000 pneumococcal genomes sequenced by the United Kingdom Health Security Agency. Experimentally, the new Xisco and SP2020 assays were compared to published assays for and . The specificity was 100% (95% CI, 98.7–100%) across all assays. The sensitivity was 100% (95% CI, 98.5–100%) for , SP2020_new and the Xisco assays and 99.6% (95% CI, 97.8–100%) for . The new assays were found to be highly sensitive and specific and able to detect as few as two pneumococcal genome copies per quantitative PCR reaction. Overall, this study demonstrated the value of performing large-scale genomic analyses of diagnostic targets, followed by testing that was specifically designed to account for global pneumococcal population-level diversity.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): molecular diagnostics , non-pneumococcal Streptococcus species , qPCR , rapid diagnostic tests , Streptococcus pneumoniae and Xisco
Funding
This study was supported by the:
  • National Institute for Health Research (Award PR-OD-1017-20007)
    • Principal Award Recipient: MartinCJ Maiden
  • Wellcome Trust (Award 218205/Z/19/Z)
    • Principal Award Recipient: AngelaBrueggemann
© 2025 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.
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2025-06-09
2026-03-10

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References

  1. Kapatai G, Sheppard CL, Al-Shahib A, Litt DJ, Underwood AP et al. Whole genome sequencing of Streptococcus pneumoniae: development, evaluation and verification of targets for serogroup and serotype prediction using an automated pipeline. PeerJ 2016; 4:e2477 [View Article] [PubMed]
     [Google Scholar]
  2. Sheppard CL, Groves N, Andrews N, Litt DJ, Fry NK et al. The genomics of Streptococcus pneumoniae carriage isolates from UK children and their household contacts, pre-PCV7 to post-PCV13. Genes 2019; 10:687 [View Article] [PubMed]
     [Google Scholar]
  3. Tuomanen EI, Mitchell TJ, Morrison DA, Spratt BG. The Pneumococcus ASM Press; 2004
     [Google Scholar]
  4. Jensen A, Scholz CFP, Kilian M. Re-evaluation of the taxonomy of the Mitis group of the genus Streptococcus based on whole genome phylogenetic analyses, and proposed reclassification of Streptococcus dentisani as Streptococcus oralis subsp. dentisani comb. nov., Streptococcus tigurinus as Streptococcus oralis subsp. tigurinus comb. nov., and Streptococcus oligofermentans as a later synonym of Streptococcus cristatus. Int J Syst Evol Microbiol 2016; 66:4803–4820 [View Article] [PubMed]
     [Google Scholar]
  5. Satzke C, Turner P, Virolainen-Julkunen A, Adrian PV, Antonio M et al. Standard method for detecting upper respiratory carriage of Streptococcus pneumoniae: updated recommendations from the World Health Organization Pneumococcal Carriage Working Group. Vaccine 2013; 32:165–179 [View Article] [PubMed]
     [Google Scholar]
  6. Sadowy E, Hryniewicz W. Identification of Streptococcus pneumoniae and other Mitis streptococci: importance of molecular methods. Eur J Clin Microbiol Infect Dis 2020; 39:2247–2256 [View Article] [PubMed]
     [Google Scholar]
  7. Ikryannikova LN, Lapin KN, Malakhova MV, Filimonova AV, Ilina EN et al. Misidentification of alpha-hemolytic streptococci by routine tests in clinical practice. Infect Genet Evol 2011; 11:1709–1715 [View Article] [PubMed]
     [Google Scholar]
  8. Diallo K, Feteh VF, Ibe L, Antonio M, Caugant DA et al. Molecular diagnostic assays for the detection of common bacterial meningitis pathogens: a narrative review. EBioMedicine 2021; 65:103274 [View Article] [PubMed]
     [Google Scholar]
  9. Tavares DA, Handem S, Carvalho RJ, Paulo AC, de Lencastre H et al. Identification of Streptococcus pneumoniae by a real-time PCR assay targeting SP2020. Sci Rep 2019; 9:3285 [View Article] [PubMed]
     [Google Scholar]
  10. Carvalho M da GS, Tondella ML, McCaustland K, Weidlich L, McGee L et al. Evaluation and improvement of real-time PCR assays targeting lytA, ply, and psaA genes for detection of pneumococcal DNA. J Clin Microbiol 2007; 45:2460–2466 [View Article] [PubMed]
     [Google Scholar]
  11. Streptococcus Lab Resources and Protocols | CDC; 2021 https://www.cdc.gov/streplab/pneumococcus/resources.html
  12. Trzciński K, Bogaert D, Wyllie A, Chu MLJN, van der Ende A et al. Superiority of trans-oral over trans-nasal sampling in detecting Streptococcus pneumoniae colonization in adults. PLoS One 2013; 8:e60520 [View Article] [PubMed]
     [Google Scholar]
  13. Ganaie FA, Govindan V, Ravi Kumar KL. Standardisation and evaluation of a quantitative multiplex real-time PCR assay for the rapid identification of Streptococcus pneumoniae. Pneumonia 2015; 6:57–66 [View Article] [PubMed]
     [Google Scholar]
  14. Strålin K, Herrmann B, Abdeldaim G, Olcén P, Holmberg H et al. Comparison of sputum and nasopharyngeal aspirate samples and of the PCR gene targets lytA and Spn9802 for quantitative PCR for rapid detection of pneumococcal pneumonia. J Clin Microbiol 2014; 52:83–89 [View Article] [PubMed]
     [Google Scholar]
  15. Salvà-Serra F, Connolly G, Moore ERB, Gonzales-Siles L. Detection of “Xisco” gene for identification of Streptococcus pneumoniae isolates. Diagn Microbiol Infect Dis 2018; 90:248–250 [View Article] [PubMed]
     [Google Scholar]
  16. Downs SL, Madhi SA, van der Merwe L, Nunes MC, Olwagen CP. Optimization of a high-throughput nanofluidic real-time PCR to detect and quantify of 15 bacterial species and 92 Streptococcus pneumoniae serotypes. Sci Rep 2023; 13:4588 [View Article] [PubMed]
     [Google Scholar]
  17. Croxen MA, Lee TD, Azana R, Hoang LMY. Use of genomics to design a diagnostic assay to discriminate between Streptococcus pneumoniae and Streptococcus pseudopneumoniae. Microb Genom 2018; 4:e000175 [View Article] [PubMed]
     [Google Scholar]
  18. Brueggemann AB, Harrold CL, Rezaei Javan R, van Tonder AJ, McDonnell AJ et al. Pneumococcal prophages are diverse, but not without structure or history. Sci Rep 2017; 7:42976 [View Article] [PubMed]
     [Google Scholar]
  19. Rezaei Javan R, Ramos-Sevillano E, Akter A, Brown J, Brueggemann AB. Prophages and satellite prophages are widespread in Streptococcus and may play a role in pneumococcal pathogenesis. Nat Commun 2019; 10:4852 [View Article] [PubMed]
     [Google Scholar]
  20. Jansen van Rensburg MJ, Berger DJ, Yassine I, Shaw D, Fohrmann A et al. Development of the Pneumococcal Genome Library, a core genome multilocus sequence typing scheme, and a taxonomic life identification number barcoding system to investigate and define pneumococcal population structure. Microb Genom 2024; 10:001280 [View Article] [PubMed]
     [Google Scholar]
  21. Jolley KA, Bliss CM, Bennett JS, Bratcher HB, Brehony C et al. Ribosomal multilocus sequence typing: universal characterization of bacteria from domain to strain. Microbiology 2012; 158:1005–1015 [View Article]
     [Google Scholar]
  22. Letunic I, Bork P. Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation. Nucleic Acids Res 2021; 49:W293–W296 [View Article] [PubMed]
     [Google Scholar]
  23. Ipcress Manual | EMBL’s European Bioinformatics Institute. n.d https://www.ebi.ac.uk/about/vertebrate-genomics/software/ipcress-manual
  24. Jolley KA, Bray JE, Maiden MCJ. Open-access bacterial population genomics: BIGSdb software, the PubMLST.org website and their applications. Wellcome Open Res 2018; 3:124 [View Article] [PubMed]
     [Google Scholar]
  25. Geneious Prime version 2022.1.1. n.d https://www.geneious.com
  26. Edgar RC. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 2004; 32:1792–1797 [View Article] [PubMed]
     [Google Scholar]
  27. Youden WJ. Index for rating diagnostic tests. Cancer 1950; 3:32–35 [View Article] [PubMed]
     [Google Scholar]
  28. Coelho JM, Kapatai G, Jironkin A, Al-Shahib A, Daniel R et al. Genomic sequence investigation Streptococcus pyogenes clusters in England (2010-2015). Clin Microbiol Infect 2019; 25:96–101 [View Article] [PubMed]
     [Google Scholar]
  29. Chalker V, Jironkin A, Coelho J, Al-Shahib A, Platt S et al. Scarlet fever incident management team. Genome analysis following a national increase in scarlet fever in england 2014. BMC Genomics 2017; 18:224 [View Article]
     [Google Scholar]
  30. Badell E, Guillot S, Tulliez M, Pascal M, Panunzi LG et al. Improved quadruplex real-time PCR assay for the diagnosis of diphtheria. J Med Microbiol 2019; 68:1455–1465 [View Article] [PubMed]
     [Google Scholar]
  31. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 2009; 55:611–622 [View Article] [PubMed]
     [Google Scholar]
  32. Buchan BW, Ledeboer NA. Emerging technologies for the clinical microbiology laboratory. Clin Microbiol Rev 2014; 27:783–822 [View Article] [PubMed]
     [Google Scholar]
  33. Fournier PE, Dubourg G, Raoult D. Clinical detection and characterization of bacterial pathogens in the genomics era. Genome Med 2014; 6:114 [View Article] [PubMed]
     [Google Scholar]
  34. Didelot X, Bowden R, Wilson DJ, Peto TEA, Crook DW. Transforming clinical microbiology with bacterial genome sequencing. Nat Rev Genet 2012; 13:601–612 [View Article] [PubMed]
     [Google Scholar]
  35. Jansen van Rensburg MJ, Swift C, Cody AJ, Jenkins C, Maiden MCJ. Exploiting bacterial whole-genome sequencing data for evaluation of diagnostic assays: campylobacter species identification as a case study. J Clin Microbiol 2016; 54:2882–2890 [View Article] [PubMed]
     [Google Scholar]
/content/journal/mgen/10.1099/mgen.0.001418
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Utilizing large and diverse bacterial genome datasets to improve the detection and identification of Streptococcus pneumoniae via PCR-based diagnostics
M Gen 11, 001418 (2025); https://doi.org/10.1099/mgen.0.001418
/content/journal/mgen/10.1099/mgen.0.001418
/content/journal/mgen/10.1099/mgen.0.001418
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