Detection of toxin-producing Corynebacterium diphtheriae from throat swabs of diphtheria patients using duplex real-time PCR

  • Yeva Rosana Mail Department of Microbiology, School of Medicine, Universitas Indonesia, Ciptomangunkusumo Hospital, Jakarta, Indonesia
  • Luh Inta Prilandari Department of Microbiology, Clinical Microbiologist Program, School of Medicine, Universitas Indonesia, Jakarta, Indonesia
  • Ruhsyahadati Ajisman Department of Microbiology, Clinical Microbiologist Program, School of Medicine, Universitas Indonesia, Jakarta, Indonesia
  • Teguh Sarry Hartono Department of Microbiology, Sulianti Saroso Infectious Diseases Hospital, Jakarta, Indonesia
  • Andi Yasmon Department of Microbiology, School of Medicine, Universitas Indonesia, Ciptomangunkusumo Hospital, Jakarta, Indonesia
Corynebacterium diphtheriae;, Diphtheria toxin;, Real-time polymerase chain reaction


Background and Objectives: Diphtheria is a potentially fatal disease caused by toxigenic bacterial infection, particularly from Corynebacterium diphtheriae (C. diphtheriae). Isolation of C. diphtheriae is technically lacking in sensitivity, and Elek’s test to detect toxin production has several difficulties associated with its application. Duplex real-time PCR to throat swab of suspected diphtheria patients can detect both bacteria and toxin-encoding genes simultaneously, faster, with higher sensitivity and specificity.
Materials and Methods: A total of 89 consecutive throat swabs from suspected diphtheria patients were collected from Sulianti Saroso Infectious Disease Hospital, Jakarta, during 2018 to 2019. Two pairs of primers and probes, targeting the rpoB gene of C. diphtheriae and the A-subunit of the diphtheria toxin gene, were used in this study. Parameters including annealing temperature, concentration of primers and probes, inhibitors, cross-reaction and detection limit were all optimized. Elek’s toxigenicity test and clinical data were analyzed for comparison.
Results: The optimum annealing temperature was 55°C. The concentrations of Cd primer, Tox primer, Cd probe and Tox probe were 0.4, 0.6, 0.5 and 0.625 µM, respectively. DNA elution and template volumes were 50 µL and 5 µL. The detection limit was 2 CFU/mL. No cross-reaction with other microorganisms was observed. Of the 89 samples, duplex real-time PCR gave better results than the standard test, with 19 (21.3%) and 10 (11.2%) patients diagnosed with diphtheria, respectively.
Conclusion: Duplex real-time PCR increases the rate of laboratory diagnosis of diphtheria, compared to the standard method to detect potentially toxigenic C. diphtheriae.


1. Leber A (2016). Clinical microbiology procedures handbook. 4th ed. Washington: ASM Press. pp: 390-399.
2. De Zoysa A, Efstratiou A, Mann G, Harrison TG, Fry NK. Development, validation and implementation of a quadruplex real-time PCR assay for identification of potentially toxigenic Corynebacteria. J Med Microbiol 2016; 65:1521-1527.
3. Clarke, Kristie. Review of the epidemiology of diphtheria 2000-2016. US Centre Disease Control Prevention. 2016.
4. Kementerian Kesehatan Republik Indonesia. Data dan informasi profil kesehatan Indonesia 2018.
5. Engler KH, Glushkevich T, Mazurova IK, George RC, Efstratiou A. A modified elek test for detection of toxigenic Corynebacteria in the diagnostic laboratory. J Clin Microbiol 1997;35:495-498.
6. Grunenwald H (2003). Optimization of polymerase chain reaction. In: Methods in molecular biology vol 226; PCR protocols. 2nd edition. Humana Press Inc. pp: 89-100.
7. Innis M, Gelfand D. Optimization of PCRs (1990). In: PCR Protocol, a guide to methods and applications. San Diego, CA: Academic Press, Inc. pp: 3-12.
8. De Zoysa A, Efstratiou A, Hawkey PM. Molecular characterization of diphtheria toxin repressor (dtxR) genes present in nontoxigenic Corynebacterium diphtheriae strains isolated in the United Kingdom. J Clin Microbiol 2005;43:223-228.
9. Pimenta FP, Matias GAM, Pereira GA, Camello TCF, Alves GB, Rosa ACP, et al. A PCR for dtxR gene: Application to diagnosis of non-toxigenic and toxigenic Corynebacterium diphtheriae. Mol Cell Probes 2008;22:189-192.
10. Sunarno, Mulyastuti Y, Puspandari N, Sariadji K. DNA sequence analysis of dtxR gene (partial) of Corynebacterium diphtheriae causing diphtheria in Jawa and Kalimantan Islands, Indonesia. Indones Biomed J 2017;9:91.
11. Khamis A, Raoult D, La Scola B. Comparison between rpoB and 16S rRNA gene sequencing for molecular identification of 168 clinical isolates of Corynebacterium. J Clin Microbiol 2005;43:1934-1936.
12. Hauser D, Popoff MR, Kiredjian M, Boquet P, Bimet F. Polymerase chain reaction assay for diagnosis of potentially toxinogenic Corynebacterium diphtheriae strains: Correlation with ADP-ribosylation activity assay. J Clin Microbiol 1993;31:2720-2723.
13. Pallen MJ, Hay AJ, Puckey LH, Efstratiou A. Polymerase chain reaction for screening clinical isolates of Corynebacteria for the production of diphtheria toxin. J Clin Pathol 1994;47:353-356.
14. Mikhailovich VM, Melnikov VG, Mazurova IK, Wachsmuth IK, Wenger JD, Wharton M, et al. Application of PCR for detection of toxigenic Corynebacterium diphtheriae strains isolated during the Russian diphtheria epidemic, 1990 through 1994. J Clin Microbiol 1995;33:3061-3063.
15. Nakao H, Pruckler JM, Mazurova IK, Narvskaia OV, Glushkevich T, Marijevski VF, et al. Heterogeneity of diphtheria toxin gene, tox, and its regulatory element, dtxR, in Corynebacterium diphtheriae strains causing epidemic diphtheria in Russia and Ukraine. J Clin Microbiol 1996;34:1711-1716.
16. Efstratiou A, Engler KH, Mazurova IK, Glushkevich T, Vuopio-Varkila J, Popovic T. Current approaches to the laboratory diagnosis of Diphtheria. J Infect Dis 2000; 181 Suppl 1:S138-145.
17. Debode F, Marien A, Janssen É, Berben G, Bragard C. The influence of amplicon length on real-time PCR results. Biotechnol Agron Soc Environ 2017;21:3-11.
18. Galazka A. The changing epidemiology of Diphtheria in the vaccine Era. J Infect Dis 2000;181 Suppl 1:S2-9.
19. Völzke H, Kloker KM, Kramer A, Guertler L, Dören M, Baumeister SE, et al. Susceptibility to diphtheria in adults: prevalence and relationship to gender and social variables. Clin Microbiol Infect 2006;12:961-967.
20. Efstratiou A, George RC. Screening tests for the presumptive identification of Corynebacterium diphtheriae in a diagnostic laboratory. J Clin Microbiol 1997; 34:3251.
21. De Zoysa A, Hawkey P, Charlett A, Efstratiou A. Comparison of four molecular typing methods for characterization of Corynebacterium diphtheriae and determination of transcontinental spread of C. diphtheriae based on BstEII rRNA gene profiles. J Clin Microbiol 2008;46:3626-3635.
22. Benamrouche N, Hasnaoui S, Badell E, Guettou B, Lazri M, Guiso N, et al. Microbiological and molecular characterization of Corynebacterium diphtheriae isolated in Algeria between 1992 and 2015. Clin Microbiol Infect 2016;22:1005.e1-1005.e7.
23. Mothershed EA, Cassiday PK, Pierson K, Mayer LW, Popovic T. Development of a real-time fluorescence PCR assay for rapid detection of the Diphtheria toxin gene. J Clin Microbiol 2002;40:4713-4719.
24. Truelove SA, Keegan LT, Moss WJ, Chaisson LH, Macher E, Azman AS, et al. Clinical and epidemiological aspects of diphtheria: a systematic review and pooled analysis. Clin Infect Dis 2020;71:89-97.
25. World Health Organization. Diphtheria vaccine: review of evidence on vaccine effectiveness and immunogenicity to assess the duration of protection ≥10 years after the last booster dose. 2017.
26. Anggraeni N, Dian PY, Umar AN, Mazanova D, Handini S, Widodo NH, et al. Pedoman Pencegahan dan Pengendalian Difteri. 2017. 1-51.
27. Weinberger B. Adult vaccination against tetanus and diphtheria: the European perspective. Clin Exp Immunol 2017;187:93-99.
28. Efstratiou A, Engler KH, Dawes CS, Sesardic D. Comparison of phenotypic and genotypic methods for detection of diphtheria toxin among isolates of pathogenic Corynebacteria. J Clin Microbiol 1998;36:3173-3177.
How to Cite
Rosana Y, Prilandari L, Ajisman R, Hartono T, Yasmon A. Detection of toxin-producing Corynebacterium diphtheriae from throat swabs of diphtheria patients using duplex real-time PCR. Iran J Microbiol. 12(6):508-515.
Original Article(s)