Document Type : Original Research


1 Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran

2 Department of Microbiology, Tonekabon Branch, Islamic Azad University, Tonekabon, Iran

3 Department of Pediatrics, Imam Khomeini Hospital, School of Medicine, Tehran University of Medical Science, Tehran, Iran

4 Department of Mycobacteriology and Pulmonary Research, Microbiology Research Center, Pasteur Institute of Iran, Tehran, Iran


Background & Objective: Streptococcus pneumoniae, Haemophilus influenzae and Streptococcus pyogenes are among the most important causes of infection in human. Inventing rapid methods to identify these species can help in providing appropriate and effective treatment options. Therefore, the current study aimed to develop a multiplex touch-down PCR method to identify rapidly the aforementioned species patients' sputum samples, simultaneously.
Methods: A total of 50 sputum samples of patients with respiratory infections resistant to treatment were collected. After DNA extraction and primer design, the complete capsule (CAP) region II, capsular polysaccharide biosynthesis (cpsA) and the structural regulator of transcription (spy) genes were amplified for detecting H. influenzae, S. pneumoniae and S. pyogenes by multiplex touch-down PCR.
Results: Among 50 samples prepared from patients with different diseases, 27 samples were positive for amplified genes. The frequency of presence of pathogens in the collected samples included 14% H. influenzae, 20% S. pneumoniae and 20% S. pyogenes. Also, in some patients, the simultaneous presence of two or three pathogens were observed.
Conclusion: In general, it can be concluded that the PCR touchdown method developed in the present study is an effective and fast method for the simultaneous identification of H. influenzae, S. pneumoniae and S. pyogenes pathogens in clinical samples of patients.


Main Subjects

  1. Loughran AJ, Orihuela CJ, Tuomanen EI. Streptococcus pneumoniae: Invasion and Inflammation. Microbiol Spectr. 2019;7(2):7.2. 15. [DOI:10.1128/microbiolspec.GPP3-0004-2018] [PMID]
  2. Walker CLF, Rudan I, Liu L, Nair H, Theodoratou E, Bhutta ZA, et al. Global burden of childhood pneumonia and diarrhoea. Lancet. 2013;381(9875):1405-16. [DOI:10.1016/S0140-6736(13)60222-6] [PMID]
  3. Brandileone MC, Casagrande ST, Guerra MLS, Zanella RC, Andrade ASS, Fabio JD. Increase in numbers of beta-lactam-resistant invasive Streptococcus pneumoniae in Brazil and the impact of conjugate vaccine coverage. J Med Microbiol. 2006;55(Pt 5):567-74. [DOI:10.1099/jmm.0.46387-0] [PMID]
  4. Avire NJ, Whiley H, Ross K. A Review of Streptococcus pyogenes: Public Health Risk Factors, Prevention and Control. Pathogens [Internet]. 2021; 10(2). [DOI:10.3390/pathogens10020248] [PMID]
  5. Wen S, Feng D, Chen D, Yang L, Xu Z. Molecular epidemiology and evolution of Haemophilus influenzae. Infect Genet Evol. 2020;80:104205. [DOI:10.1016/j.meegid.2020.104205] [PMID]
  6. Takeuchi N, Ohkusu M, Hoshino T, Yamamoto S, Segawa S, Murata S, et al. Emergence of Haemophilus influenzae with low susceptibility to quinolones isolated from pediatric patients in Japan. J Infect Chemother. 2021;27(7):1020-6. [DOI:10.1016/j.jiac.2021.02.022] [PMID]
  7. Marik PE. Aspiration pneumonitis and aspiration pneumonia. N Engl J Med. 2001;344(9):665-71. [DOI:10.1056/NEJM200103013440908] [PMID]
  8. Hartl D, Tirouvanziam R, Laval J, Greene CM, Habiel D, Sharma L, et al. Innate Immunity of the Lung: From Basic Mechanisms to Translational Medicine. J Innate Immun. 2018;10(5-6):487-501. [DOI:10.1159/000487057] [PMID]
  9. Boyles T, Wasserman S. Diagnosis of bacterial infection. S Afr Med J. 2015;105(5). [DOI:10.7196/SAMJ.9647]
  10. Trotter AJ, Aydin A, Strinden MJ, O'Grady J. Recent and emerging technologies for the rapid diagnosis of infection and antimicrobial resistance. Curr Opin Microbiol. 2019;51:39-45. [DOI:10.1016/j.mib.2019.03.001] [PMID]
  11. Gerace E, Mancuso G, Midiri A, Poidomani S, Zummo S, Biondo C. Recent Advances in the Use of Molecular Methods for the Diagnosis of Bacterial Infections. Pathogens. 2022;11(6):663. [DOI:10.3390/pathogens11060663] [PMID]
  12. Ferrera I, Balagué V, Voolstra CR, Aranda M, Bayer T, Abed RM, et al. Molecular methods for biofilms. Biofouling Methods. 2014:87-137. [DOI:10.1002/9781118336144.ch4]
  13. Goodarzi NN, Pourmand M, Rajabpour M, Arfaatabar M, Mosadegh M, Mohamad SS. Frequency of Mycoplasma pneumoniae, Legionella pneumophila and Chlamydia spp. among patients with atypical pneumonia in Tehran. New Microbes New Infect. 2020;37:100744. [DOI:10.1016/j.nmni.2020.100744] [PMID]
  14. Lim HJ, Kang ER, Park MY, Kim BK, Kim MJ, Jung S, et al. Development of a multiplex real-time PCR assay for the simultaneous detection of four bacterial pathogens causing pneumonia. PLoS One. 2021;16(6):e0253402. [DOI:10.1371/journal.pone.0253402] [PMID]
  15. Maleki A, Mansournia F, Ghafourian S, Taherikalani M, Pakzad I, Mohammadi J, et al. Rapid and direct molecular detection of Streptococcus pneumoniae and Haemophilus influenzae isolated in oropharynx and nasal cavity of children. New Microbes New Infect. 2020;33:100632. [DOI:10.1016/j.nmni.2019.100632] [PMID]
  16. Thors V, Morales-Aza B, Pidwill G, Vipond I, Muir P, Finn A. Population density profiles of nasopharyngeal carriage of 5 bacterial species in pre-school children measured using quantitative PCR offer potential insights into the dynamics of transmission. Hum Vaccin Immunother. 2016;12(2):375-82. [DOI:10.1080/21645515.2015.1090069] [PMID]
  17. Gillis HD, Lang ALS, ElSherif M, Martin I, Hatchette TF, McNeil SA, et al. Assessing the diagnostic accuracy of PCR-based detection of Streptococcus pneumoniae from nasopharyngeal swabs collected for viral studies in Canadian adults hospitalised with community-acquired pneumonia: a Serious Outcomes Surveillance (SOS) Network of the Canadian Immunization Research (CIRN) study. BMJ Open. 2017;7(6):e015008. [DOI:10.1136/bmjopen-2016-015008] [PMID]
  18. Bjarnason A, Lindh M, Westin J, Andersson LM, Baldursson O, Kristinsson KG, et al. Utility of oropharyngeal real-time PCR for S. pneumoniae and H. influenzae for diagnosis of pneumonia in adults. Eur J Clin Microbiol Infect Dis. 2017;36(3):529-36. [DOI:10.1007/s10096-016-2829-z] [PMID] []
  19. Shakib P, Zolfaghari MR. Detection of Streptococcus pneumoniae in sputum samples by real-time PCR. Anti-Infect Agents. 2021;19(1):117-21. [DOI:10.2174/2211352518999200629165108]
  20. Fan X, Liu X, Ji L, Cai D, Jiang J, Zhu J, et al. Epidemiological analysis and rapid detection by one-step multiplex PCR assay of Haemophilus influenzae in children with respiratory tract infections in Zhejiang Province, China. BMC Infect Dis. 2018;18(1):414. [DOI:10.1186/s12879-018-3295-2] [PMID] []
  21. Aydemir O, Aydemir Y, Ozdemir M. The role of multiplex PCR test in identification of bacterial pathogens in lower respiratory tract infections. Pak J Med Sci. 2014;30(5):1011-6. [DOI:10.12669/pjms.305.5098] [PMID]
  22. Gadsby NJ, Russell CD, McHugh MP, Mark H, Conway Morris A, Laurenson IF, et al. Comprehensive Molecular Testing for Respiratory Pathogens in Community-Acquired Pneumonia. Clin Infect Dis. 2016;62(7):817-23. [DOI:10.1093/cid/civ1214] [PMID] []
  23. Naderi H, Sheybani F, Sarvghad M, Meshkat Z, Jabbari Nooghabi M. Etiological Diagnosis of Community-Acquired Pneumonia in Adult Patients: A Prospective Hospital-Based Study in Mashhad, Iran. Jundishapur J Microbiol. 2015;8(8):e22780. [DOI:10.5812/jjm.22780] [PMID] []
  24. Temesgen D, Bereded F, Derbie A, Biadglegne F. Bacteriology of community acquired pneumonia in adult patients at Felege Hiwot Referral Hospital, Northwest Ethiopia: a cross-sectional study. Antimicrob Resist Infect Control. 2019;8(1):1-8. [DOI:10.1186/s13756-019-0560-0] [PMID] []
  25. Wang Y, Kong F, Yang Y, Gilbert GL. A multiplex PCR-based reverse line blot hybridization (mPCR/RLB) assay for detection of bacterial respiratory pathogens in children with pneumonia. Pediatr Pulmonol. 2008;43(2):150-9. [DOI:10.1002/ppul.20749] [PMID]
  26. Park HK, Lee S-J, Yoon JW, Shin JW, Shin H-S, Kook J-K, et al. Identification of the cpsA gene as a specific marker for the discrimination of Streptococcus pneumoniae from viridans group streptococci. J Med Microbiol. 2010;59(10):1146-52. [DOI:10.1099/jmm.0.017798-0] [PMID]
  27. Park HK, Lee S-J, Yoon JW, Shin JW, Shin H-S, Kook J-K, et al. Identification of the cpsA gene as a specific marker for the discrimination of Streptococcus pneumoniae from viridans group streptococci. J Med Microbiol. 2010;59(10): 1146-52. [DOI:10.1099/jmm.0.017798-0]
  28. Asgari A, Ataee R, Tavana A M, Mirnejad R, Ghorbanalizdgan M. Real-time RT PCR Evaluation of the Xylitol Effect on the Expression of Streptococcus pneumoniae cpsB,cpsD and psaA Genes. Iran J Med Microbiol 2021; 15 (4) :392-399. [DOI: 10.30699/ijmm.15.4.392]