Eubacterial PCR - the usefulness of the molecular method in clinical practice
Abstract
Abstract
Background: Isolation and identification of bacterial pathogens enables accurate diagnosis of bacterial infection, allowing rational use of appropriate narrow-spectrum antibiotics. In some cases, the routine bacterial culture can give negative results. In those cases additional use of molecular techniques such as eubacterial (broad-range 16S rRNA) PCR may detect and identify bacterial genetic material.
Methods: Between February 2012 and April 2013 42 specimens from 35 patients, already treated with antimicrobials, were taken and tested by eubacterial PCR in addition to routine microbiological culture. Results: Eubacterial PCR yielded positive result in 21/42 specimens in 18 patients (in three mixed sequences). Therefore, in 15 patients the diagnosis of bacterial infection was obtained with DNA identification and the results were interpreted in accordance to patients’ history, laboratory and image diagnostics. Only 4 specimens were culture-positive.
Conclusions: Although eubacterial PCR enables the identification of any bacterial DNA in clinical specimens, there are some limitations: no information concerning antimicrobial susceptibility of the causative agents, problem of differentiating living from dead bacteria and problem to differentiate contaminants from pathogenic bacteria. The method is also expensive. In the following article recommendations for appropriate and rational use of eubacterial PCR are presented.
Downloads
References
Pulcini C, Gyssens IC. How to educate prescribers in antimicrobial stewardship practices.Virulence. 2013; 4(2): 192-202.
Harris KA in Hartley JC. Development of broad-range 16S rDNA PCR for use in the routine diagnostic clinical microbiology service. J Med Microbiol. 2003; 52: 685-91.
Sibley CD, Peirano G, Church DL. Molecular methods for pathogen and microbial community detection and characterization: current and potential application in diagnostic microbiology. Infect Genet Evol. 2012; 12(3): 505-21.
Sontakke S, Cadenas MB, Maggi RG, Diniz PP, Breitschwerdt EB. Use of broad range 16S rDNA PCR in clinical microbiology. J Microbiol Methods. 2009; 76: 217-25.
Jensen KH, Dargis R, Christensen JJ, Kemp M. Ribosomal PCR and DNA sequencing for detection and identification of bacteria: experience from 6 years of routine analyses of patient samples. APMIS. 2013 Jul 24.
Mignard S, Flandrois JP. 16S rRNA sequencing in routine bacterial identification: a 30-month experiment. J Microbiol Methods. 2006; 67: 574-81.
Doolittle WF. Phylogenetic classification and the universal tree. Science. 1999; 284: 2124-8.
Rantakokko-Jalava K, Nikkari S, Jalava J, Eerola E, Skurnik M, Meurman O, et al. Direct amplification of rRNA genes in diagnosis of bacterial infections. J Clin Microbiol. 2000; 38(1): 32-9.
Bosshard PP, Kronenberg A, Zbinden R, Ruef C, Böttger EC in Altwegg M. Etiologic Diagnosis of Infective Endocarditis by Broad-Range Polymerase Chain Reaction: A 3-Year Experience. Clin Infect Dis. 2003; 37(2):167–72.
Kommedal O, Lekang K, Langeland N, Wiker HG. Characterization of polybacterial clinical samples using a set of group-specific broad-range primers targeting the 16S rRNA gene followed by DNA sequencing and RipSeq analysis. J Med Microbiol. 2011; 60(Pt 7): 927-36.
Döring G, Unertl K, Heininger A. Validation criteria for nucleic acid amplification techniques for bacterial infections. Clin Chem Lab Med. 2008; 46(7): 909-18.
Welinder-Olsson C, Dotevall L, Hogevik H, Jungnelius R, Trollfors B, Wahl M, et al. Comparison of broad-range bacterial PCR and culture of cerebrospinal fluid for diagnosis of community-acquired bacterial meningitis. Clin Microbiol Infect. 2007; 13(9): 879-86.
Rosey AL, Abachin E, Quesnes G, Cadilhac C, Pejin Z, Glorion C, et al. Development of a broad-range 16S rDNA real-time PCR for the diagnosis of septic arthritis in children. J Microbiol Methods. 2007; 68(1): 88-93.
Ammann RA, Zucol F, Aebi C, Niggli FK, Kühne T, Nadal D. Real-time broad-range PCR versus blood culture. A prospective pilot study in pediatric cancer patients with fever and neutropenia. Support Care Cancer. 2007; 15(6): 637-41.
The Author transfers to the Publisher (Zdravniški vestnik/Slovenian Medical Journal) all economic copyrights following form Article 22 of the Slovene Copyright and Related Rights Act (ZASP), including the right of reproduction, the right of distribution, the rental right, the right of public performance, the right of public transmission, the right of public communication by means of phonograms and videograms, the right of public presentation, the right of broadcasting, the right of rebroadcasting, the right of secondary broadcasting, the right of communication to the public, the right of transformation, the right of audiovisual adaptation and all other rights of the author according to ZASP.
The aforementioned rights are transferred non-exclusively, for an unlimited number of editions, for the term of the statutory
The Author can make use of his work himself or transfer subjective rights to others only after 3 months from date of first publishing in the journal Zdravniški vestnik/Slovenian Medical Journal.
The Publisher (Zdravniški vestnik/Slovenian Medical Journal) has the right to transfer the rights, acquired parties without explicit consent of the Author.
The Author consents that the Article be published under the Creative Commons BY-NC 4.0 (attribution-non-commercial) or comparable licence.
