Molecular Biotechnology

, Volume 42, Issue 1, pp 117–127

Molecular Detection of Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis

  • Erica D. Dawson
  • Amber W. Taylor
  • James A. Smagala
  • Kathy L. Rowlen


We developed molecular diagnostic assays for the detection of Streptococcus pyogenes (GAS) and Streptococcus dysgalactiae subsp. equisimilis (SDSE), two streptococcal pathogens known to cause both pharyngitis and more invasive forms of disease in humans. Two real-time PCR assays coupled with an internal control were designed to be performed in parallel. One assay utilizes a gene target specific to GAS, and the other utilizes a gene target common to the two species. Both assays showed 2–3 orders of magnitude improved analytical sensitivity when compared to a commercially available rapid antigen test. In addition, when compared to standard culture in an analysis of 96 throat swabs, the real-time PCR assays resulted in clinical sensitivity and specificity of 91.7 and 100%, respectively. As capital equipment costs for real-time PCR can be prohibitive in smaller laboratories, the real-time PCR assays were converted to a low-density microarray format designed to function with an inexpensive photopolymerization-based non-enzymatic signal amplification (NESA™) method. S. pyogenes was successfully detected on the low-density microarray in less than 4 h from sample extraction through detection.


Streptococcus pyogenes Streptococcus dysgalactiae subspecies equisimilis Real-time PCR Low-density microarray Molecular diagnostics Pathogen detection 


  1. 1.
    Ferretti, J. J., McShan, W. M., Ajdic, D., Savic, D. J., Savic, G., Lyon, K., et al. (2001). Complete genome sequence of an M1 strain of Streptococcus pyogenes. Proceedings of the National Academy of Sciences of the United States of America, 8(98), 4658–4663. doi:10.1073/pnas.071559398.CrossRefGoogle Scholar
  2. 2.
    Todar, K. (2006). Todar’s online textbook of bacteriology: Streptococcus pyogenes. Accessed 26 June 2007.
  3. 3.
    Humair, J.-P., Revaz, S. A., Bovier, P. I., & Stalder, H. (2006). Management of acute pharyngitis in adults: Reliability of rapid streptococcal tests and clinical findings. Archives of Internal Medicine, 166, 640–644. doi:10.1001/archinte.166.6.640.CrossRefGoogle Scholar
  4. 4.
    Snow, V., Mottur-Pilson, C., Cooper, R. J., & Hoffman, J. R. (2001). Principles of appropriate antibiotic use for acute pharyngitis in adults. Annals of Internal Medicine, 134(6), 506–508.Google Scholar
  5. 5.
    Bourbeau, P. P. (2003). Role of the microbiology laboratory in diagnosis and management of pharyngitis. Journal of Clinical Microbiology, 41(8), 3467–3472. doi:10.1128/JCM.41.8.3467-3472.2003.CrossRefGoogle Scholar
  6. 6.
    Davies, M. R., McMillan, D. J., Beiko, R. G., Barroso, V., Geffers, R., Sriprakash, K. S., et al. (2007). Virulence profiling of Streptococcus dysgalactiae subspecies equisimilis isolated from infected humans reveals 2 distinct genetic lineages that do not segregate with their phenotypes or propensity to cause diseases. Clinical Infectious Diseases, 44, 1442–1454. doi:10.1086/516780.CrossRefGoogle Scholar
  7. 7.
    Cohen-Poradosu, R., Jaffe, J., Lavi, D., Grisariu-Greenzaid, S., Nir-Paz, R., Valinsky, L., et al. (2004). Group G streptococcal bacteremia in Jerusalem. Emerging Infectious Diseases, 10(8), 1455–1460.Google Scholar
  8. 8.
    Dierksen, K. P., & Tagg, J. R. (2000). Haemolysin-deficient variants of Streptococcus pyogenes and S. dysgalactiae subsp. equisimilis may be overlooked as aetiological agents of pharyngitis. Journal of Medical Microbiology, 49, 811–816.Google Scholar
  9. 9.
    Fox, K., Turner, J., & Fox, A. (1993). Role of beta-hemolytic group C streptococci in pharyngitis: Incidence and biochemical characteristics of Streptococcus equisimilis and Streptococcus anginosus in patients and healthy controls. Journal of Clinical Microbiology, 31(4), 804–807.Google Scholar
  10. 10.
    Gerber, M. A., & Schulman, S. T. (2004). Rapid diagnosis of pharyngitis caused by group A streptococci. Clinical Microbiology Reviews, 17(3), 571–580. doi:10.1128/CMR.17.3.571-580.2004.CrossRefGoogle Scholar
  11. 11.
    Lewis, R. F. M., & Balfour, A. E. (1999). Group C streptococci isolated from throat swabs: A laboratory and clinical study. Journal of Clinical Pathology, 52(4), 264–266.CrossRefGoogle Scholar
  12. 12.
    Lindbæk, M., Høiby, E. A., Lermark, G., Steinsholt, I. M., & Hjortdahl, P. (2005). Clinical symptoms and signs in sore throat patients with large colony variant ß-haemolytic streptococci groups C or G versus group A. The British Journal of General Practice, 55, 615–619.Google Scholar
  13. 13.
    Turner, J. C., Hayden, F. G., Lobo, M. C., Ramirez, C. E., & Murren, D. (1997). Epidemiologic evidence for Lancefield group C beta-hemolytic streptococci as a cause of exudative pharyngitis in college students. Journal of Clinical Microbiology, 35(1), 1–4.Google Scholar
  14. 14.
    Turner, J. C., Fox, A., Fox, K., Addy, C., Garrison, C. Z., Herron, B., et al. (1993). Role of beta-hemolytic group C streptococci in pharyngitis: Epidemiological study of clinical features associated with isolation of group C streptococci. Journal of Clinical Microbiology, 31(4), 808–811.Google Scholar
  15. 15.
    Zaoutis, T., Attia, M., Gross, R., & Klein, J. (2004). The role of group C and group G streptococci in acute pharyngitis in children. Clinical Microbiology & Infection, 10(1), 37–40. doi:10.1111/j.1469-0691.2004.00732.x.CrossRefGoogle Scholar
  16. 16.
    Facklam, R. (2002). What happened to the streptococci: Overview of taxonomic and nomenclature changes. Clinical Microbiology Reviews, 15(4), 613–630. doi:10.1128/CMR.15.4.613-630.2002.CrossRefGoogle Scholar
  17. 17.
    Baracco, G. J., & Bisno, A. L. (2006). Group C and Group G streptococcal infections: Epidemiologic and clinical aspects. In V. A. Fischetti, R. P. Novick, J. J. Ferretti, D. A. Portnoy, & J. I. Rood (Eds.), Gram-positive pathogens (2nd ed.). Washington, DC: American Society for Microbiology.Google Scholar
  18. 18.
    Efstratiou, A. (1997). Pyogenic streptococci of Lancefield Groups C and G as pathogens in man. Journal of Applied Microbiology, 83(Suppl 1), 72S–79S. doi:10.1046/j.1365-2672.83.s1.8.x.CrossRefGoogle Scholar
  19. 19.
    Stevens, D. L. (2000). Streptococcal toxic shock syndrome associated with necrotizing fasciitis. Annual Review of Medicine, 51, 271–288. doi:10.1146/ Scholar
  20. 20.
    Ekelund, K., Skinhøj, P., Madsen, J., & Konradsen, H. B. (2005). Invasive Group A, B, C, and G Streptococcal infections in Denmark 1999–2002: Epidemiological and clinical aspects. Clinical Microbiology & Infection, 11(7), 569–576. doi:10.1111/j.1469-0691.2005.01169.x.CrossRefGoogle Scholar
  21. 21.
    Health Protection Agency (UK), Microbiological Diagnostics Assessment Service. (2005). Group A streptococcus antigen detection test kits: A review of evaluation literature (04123). Accessed 28 June 2007.
  22. 22.
    Uhl, J. R., Adamson, S. C., Vetter, E. A., Schleck, C. D., Harmsen, W. S., Iverson, L. K., et al. (2003). Comparison of LightCycler PCR, rapid antigen immunoassay, and culture for detection of group A streptococci from throat swabs. Journal of Clinical Microbiology, 41(1), 242–249. doi:10.1128/JCM.41.1.242-249.2003.CrossRefGoogle Scholar
  23. 23.
    Bisno, A. L., Gerber, M. A., Gwaltney, J. M., Jr., Kaplan, E. L., & Schwartz, R. H. (2002). Practice guidelines for the diagnosis and management of group A streptococcal pharyngitis. Clinical Infectious Diseases, 35, 113–125. doi:10.1086/340949.CrossRefGoogle Scholar
  24. 24.
    Espy, M. J., Uhl, R. J., Sloan, L. M., Buchwalkter, S. P., Jones, M. F., Vetter, E. A., et al. (2006). Real-time PCR in clinical microbiology: Applications for routine laboratory testing. Clinical Microbiology Reviews, 19(1), 165–256. doi:10.1128/CMR.19.1.165-256.2006.CrossRefGoogle Scholar
  25. 25.
    Mackay, I. M., Arden, K. E., & Nitsche, A. (2002). Real-time PCR in virology. Nucleic Acids Research, 30(6), 1292–1305. doi:10.1093/nar/30.6.1292.CrossRefGoogle Scholar
  26. 26.
    Bodrossy, L., & Sessitsch, A. (2004). Oligonucleotide microarrays in microbial diagnostics. Current Opinion in Microbiology, 7(3), 245–254. doi:10.1016/j.mib.2004.04.005.CrossRefGoogle Scholar
  27. 27.
    Cleven, B. E. E., Palka-Santini, M., Gielen, J., Meembor, S., Krönke, M., & Krut, O. (2006). Identification and characterization of bacterial pathogens causing bloodstream infections by DNA microarray. Journal of Clinical Microbiology, 44(7), 2389–2397. doi:10.1128/JCM.02291-05.CrossRefGoogle Scholar
  28. 28.
    Roth, S. B., Jalava, J., Ruuskanen, O., Ruohola, A., & Nikkari, S. (2004). Use of an oligonucleotide microarray for laboratory diagnosis of bacteria responsible for acute upper respiratory infections. Journal of Clinical Microbiology, 42(9), 4268–4274. doi:10.1128/JCM.42.9.4268-4274.2004.CrossRefGoogle Scholar
  29. 29.
    Zammatteo, N., Hamels, S., DeLongueville, F., Alexandre, I., Gala, J., Brasseur, F., et al. (2002). New chips for molecular biology and diagnostics. Biotechnology Annual Review, 8, 85–101. doi:10.1016/S1387-2656(02)08005-5.CrossRefGoogle Scholar
  30. 30.
    Hall, T. A. (1999). BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series, 41, 95–98.Google Scholar
  31. 31.
    Rozen, S., & Skaletsky, H. J. (2000). Primer3 on the WWW for general users and for biologist programmers. In S. Krawetz & S. Misener (Eds.), Methods in molecular biology, vol. 132: Bioinformatics methods and protocols (pp. 365–386). Totowa, NJ: Humana Press.Google Scholar
  32. 32.
    Hoorfar, J., Abdulmawjood, A., Cook, N., Wagner, M., & Fach, P. (2004). Practical considerations in design of internal amplification controls for diagnostic PCR assays. Journal of Clinical Microbiology, 42(5), 1863–1868. doi:10.1128/JCM.42.5.1863-1868.2004.CrossRefGoogle Scholar
  33. 33.
    Kuck, L., & Taylor, A. W. (2008). Photopolymerization as an innovative detection technique for low-density microarrays. BioTechniques, 45, 179–186. doi:10.2144/000112889.CrossRefGoogle Scholar
  34. 34.
    Higuchi, R. G., & Ochman, H. (1989). Production of single-stranded DNA templates by exonuclease digestion following the polymerase chain reaction. Nucleic Acids Research, 17(14), 5865. doi:10.1093/nar/17.14.5865.CrossRefGoogle Scholar
  35. 35.
    United States Food and Drug Administration.(USFDA). (2008). Draft guidance for industry and FDA staff: Establishing the performance characteristics of in vitro diagnostic devices for the detection or detection and differentiation of influenza viruses.
  36. 36.
    Templeton, K. E., Scheltinga, S. A., Beersma, M. F. C., Kroes, A. C. M., & Claas, E. C. J. (2004). Rapid and sensitive method using multiplex real-time PCR for diagnosis of infections by influenza A and influenza B viruses, respiratory syncytial virus, and parainfluenza viruses 1, 2, 3, and 4. Journal of Clinical Microbiology, 42(4), 1564–1569. doi:10.1128/JCM.42.4.1564-1569.2004.CrossRefGoogle Scholar
  37. 37.
    Lawrence, J., Yajko, D. M., & Hadley, K. (1985). Incidence and characterization of beta-hemolytic Streptococcus milleri and differentiation from S. pyogenes (group A), S. equisimilis (group C), and large-colony group G streptococci. Journal of Clinical Microbiology, 22(5), 772–777.Google Scholar

Copyright information

© Humana Press 2009

Authors and Affiliations

  • Erica D. Dawson
    • 1
  • Amber W. Taylor
    • 1
  • James A. Smagala
    • 1
    • 2
  • Kathy L. Rowlen
    • 1
  1. 1.InDevR Inc.BoulderUSA
  2. 2.Influenza DivisionThe Centers for Disease Control and PreventionAtlantaUSA

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