The investigation of respiratory infections by molecular techniques provides important information about the epidemiology of respiratory disease, especially during the post-vaccination era. The objective of the present study was the detection of bacterial pathogens directly in clinical samples from patients with upper and lower respiratory tract infections using multiplex polymerase chain reaction (PCR) assays developed in our laboratory. Clinical samples taken over a three-year period (2007–2009) and obtained from 349 patients (adults (n = 66); children (n = 283)) with signs and symptoms of certain upper or lower respiratory tract infections, consisted of: bronchoalveolar lavages (BAL, n = 83), pleural fluids (n = 29), and middle-ear aspirates (n = 237). Overall, 212 samples (61%) were confirmed by culture and/or PCR. Among the positive samples, Streptococcus pneumoniae (mainly serotype 3) was predominant (104/212; 49.0%), followed by non-typable Haemophilus influenza e ( NTHi) 59/212; 27.8%) and Streptococcus pyogenes (47/212; 22%). Haemophilus influenzae type b was detected in only three samples. The underlying microbiology of respiratory infections is gradually changing in response to various selective pressures, such as vaccine use and antibiotic consumption. The application of multiplex PCR (mPCR) assays is particularly useful since it successfully identified the microorganisms implicated in acute otitis media or lower respiratory tract infections in nearly 75% of patients with a positive result compared to conventional cultures. Non-culture identification of the implicated pneumococcal serotypes is also an important issue for monitoring pneumococcal infections in the era of conjugate pneumococcal vaccines.
References
[1]
Garcia-Rodriguez, J.A.; Martinez, M.J.F. Dynamics of nasopharyngeal colonization by potential respiratory pathogens. J. Antimicrob. Chemother. 2002, 50, 59–73, doi:10.1093/jac/dkf506.
[2]
Faden, H.; Duffy, L.; Wasielewski, R.; Wolf, J.; Krystofik, D.; Tung, Y. Relationship between nasopharyngeal colonization and the development of otitis media in children. J. Infec. Dis. 1997, 175, 1440–1445.
[3]
Faden, H.; Duffy, L.; Williams, D.A.; Krystofik, D.; Wolf, J. Epidemiology of nasopharyngeal colonization with non-typeable H. influenzae in the first two years of life. Acta Otolaryngol. Suppl. 1996, 523, 128–129.
[4]
Wolf, J.; Daley, A.J. Microbiological aspects of bacterial lower respiratory tract illness in children: Typical pathogens. Paed. Resp. Rev. 2007, 8, 204–211, doi:10.1016/j.prrv.2007.08.002.
[5]
Cherian, T.; Lalitha, M.K.; Manoharan, A.; Thomas, K.; Yolken, R.H.; Steinhoff, M.C. PCR-enzyme immunoassay for detection of S. pneumoniae DNA in cerebrospinal fluid samples from patients with culture-negative meningitis. J. Clin. Microbiol. 1998, 36, 3605–3608.
[6]
Tzanakaki, G.; Tsolia, M.; Vlachou, V.; Theodoridou, M.; Pangalis, A.; Foustoukou, M.; Karpathios, T.; Blackwell, C.C.; Kremastinou, J. Evaluation of non-culture diagnosis of invasive meningococcal disease by polymerase chain reaction (PCR). FEMS Immunol. Med. Microbiol. 2003, 39, 31–36, doi:10.1016/S0928-8244(03)00175-5.
[7]
Tzanakaki, G.; Tsopanomichalou, M.; Kesanopoulos, K.; Matzourani, R.; Sioumala, M.; Tabaki, A.; Kremastinou, J. Simultaneous single-tube PCR assay for the detection of Neisseria meningitidis, Haemophilus influenzae type b and Streptococcus pneumoniae. Clin. Microbiol. Infect. 2005, 11, 386–390, doi:10.1111/j.1469-0691.2005.01109.x.
[8]
Xirogianni, A.; Tzanakaki, G.; Karagianni, E.; Markoulatos, P.; Kourea-Kremastinou, J. Development of a single-tube PCR assay for the simultaneous detection of Haemophilus influenzae, Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus spp. directly in clinical samples. Diagn. Microbiol. Infec. Dis. 2009, 63, 121–126, doi:10.1016/j.diagmicrobio.2008.09.017.
[9]
Sioumala, M.; Tzanakaki, G.; Kesanopoulos, K.; Levidiotou-Stefanou, S.; Kourea-Kremastinou, J. Simultaneous detection of nine serotypes of Streptococcus pneumoniae using stepdown multiplex PCR. Acta Microbiol. Hell. 2007, 52, 173–179.
[10]
Str？lin, K.; B？ckman, A.; Holmberg, H.; Fredlund, H.; Olcén, P. Design of a multiplex PCR for Streptococcus pneumoniae, Haemophilus influenzae, Mycoplasma pneumoniae and Chlamydophila pneumoniae to be used on sputum samples. APMIS 2005, 113, 99–111, doi:10.1111/j.1600-0463.2005.apm1130203.x.
[11]
Brito, D.A.; Ramirez, M.; de Lencastre, H. Serotyping Streptococcus pneumoniae by multiplex PCR. J. Clin. Microbiol. 2003, 41, 2378–2384, doi:10.1128/JCM.41.6.2378-2384.2003.
[12]
FastPCR Software. Available online: http://www.biocenter.helsinki.fi/bi/programs/fastpcr.htm (accessed on 1 February 2013).
[13]
Liu, D.; Hollingshead, S.; Swiatlo, E.; Lawrence, M.L.; Austin, F.W. Rapid identification of Streptococcus pyogenes with PCR primers from a putative transcriptional regulator gene. Res. Microbiol. 2005, 156, 564–567, doi:10.1016/j.resmic.2005.01.010.
[14]
Primer3 Software. Available online: http://frodo.wi.mit.edu (accessed on 1 February 2013).
[15]
Kong, F.; Ma, L.; Gwendolyn, G. Simultaneous detection and serotype identification of Streptococcus agalactiae using multiplex PCR and reverse line blot hybridization. J. Med. Microbiol. 2005, 54, 1133–1138, doi:10.1099/jmm.0.46244-0.
[16]
Palmu, A.A.; Saukkoriipi, P.A.; Lahdenkari, M.I.; Kuisma, L.K.; Makela, P.H.; Kilpi, T.M.; Leinonen, M. Does the presence of pneumococcal DNA in middle-ear fluid indicate pneumococcal aetiology in acute otitis media? J. Infect. Dis. 2004, 189, 775–784, doi:10.1086/381765.
[17]
Ueyama, T.; Kurono, Y.; Shirabe, K.; Takeshita, M.; Mogi, G. High incidence of Haemophilus influenzae in nasopharyngeal secretions and middle ear effusions as detected by PCR. J. Clin. Microbiol. 1995, 33, 1835–1838.
[18]
Post, J.C.; Preston, R.A.; Aul, J.J.; Larkins-Pettigrew, M.; Rydquist-White, J.; Anderson, K.W.; Wadowsky, R.M.; Reagan, D.R.; Walker, E.S.; Kingsley, L.A. Molecular analysis of bacterial pathogens in otitis media with effusion. JAMA 1995, 273, 1598–1604, doi:10.1001/jama.1995.03520440052036.
[19]
Lynch, J.P.; Zhanel, G.G. Streptococcus pneumoniae: Epidemiology and risk factors, evolution of antimicrobial resistance, and impact of vaccines. Curr. Opin. Pulm. Med. 2010, 16, 217–225.
[20]
Yamanaka, N.; Hotomi, M.; Billal, D.S. Clinical bacteriology and immunology in acute otitis media in children. J. Infect. Chemother. 2008, 14, 180–187, doi:10.1007/s10156-007-0599-3.
[21]
Vergison, A. Microbiology of otitis media: A moving target. Vaccine 2008, 26S, G5–G10, doi:10.1016/j.vaccine.2008.11.006.
[22]
Matar, G.M.; Sidani, N.; Fayad, M.; Hadi, U. Two-step PCR-based assay for identification of bacterial etiology of otitis media with effusion in infected Lebanese children. J. Clin. Microbiol. 1998, 36, 1185–1188.
[23]
Segal, N.; Givon-Lavi, N.; Leibovitz, E.; Yagupsky, P.; Leiberman, A.; Dagan, R. Acute otitis media caused by Streptococcus pyogenes in children. Clin. Infec. Dis. 2005, 41, 35–41, doi:10.1086/430605.
[24]
Stralin, K.; Korsgaard, J.; Olcen, P. Evaluation of a multiplex PCR for bacterial pathogens applied to bronchoalveolar lavage. Eur. Respir. J. 2006, 28, 568–575, doi:10.1183/09031936.06.00006106.
[25]
Carroll, K.C. Laboratory diagnosis of lower respiratory tract infections: Controversy and conundrums. J. Clin. Microbiol. 2002, 40, 3115–3120, doi:10.1128/JCM.40.9.3115-3120.2002.
[26]
Ioanas, M.; Ferrer, R.; Angrill, J.; Ferrer, M.; Torres, A. Microbial investigation in ventilator-associated pneumonia. Eur. Respir. J. 2001, 17, 791–801, doi:10.1183/09031936.01.17407910.
Li, S.-T.T.; Tancredi, D.J. Empyema hospitalizations increased in US children despite pneumococcal conjugate vaccine. Paediatrics 2010, 125, 26–33, doi:10.1542/peds.2009-0184.
[29]
Pilishvili, T.; Lexau, C.; Farley, M.M.; Hadler, J.; Harrison, L.H.; Bennett, N.M.; Reingold, A.; Thomas, A.; Schaffner, W.; Craig, A.S.; et al. Active bacterial core surveillance/emerging infections program network. Sustained reductions in invasive pneumococcal disease in the era of conjugate vaccine. J. Infect. Dis. 2010, 201, 32–41, doi:10.1086/648593.
[30]
Roxburgh, C.S.; Youngson, G.G.; Townend, J.A.; Turner, S.W. Trends in pneumonia and empyema in Scottish children in the past 25 years. Arch. Dis. Child. 2008, 93, 316–318, doi:10.1136/adc.2007.126540.
