title: Real-time PCR assays compared to culture-based approaches for identification of aerobic bacteria in chronic wounds.
abstract: Chronic wounds cause substantial morbidity and disability. Infection in chronic wounds is clinically defined by routine culture methods that can take several days to obtain a final result, and may not fully describe the community of organisms or biome within these wounds. Molecular diagnostic approaches offer promise for a more rapid and complete assessment. We report the development of a suite of real-time PCR assays for rapid identification of bacteria directly from tissue samples. The panel of assays targets 14 common, clinically relevant, aerobic pathogens and demonstrates a high degree of sensitivity and specificity using a panel of organisms commonly associated with chronic wound infection. Thirty-nine tissue samples from 29 chronic wounds were evaluated and the results compared with those obtained by culture. As revealed by culture and PCR, the most common organisms were methicillin-resistant Staphylococcus aureus (MRSA) followed by Streptococcus agalactiae (Group B streptococcus) and Pseudomonas aeruginosa. The sensitivities of the PCR assays were 100% and 90% when quantitative and qualitative culture results were used as the reference standard, respectively. The assays allowed the identification of bacterial DNA from ten additional organisms that were not revealed by quantitative or qualitative cultures. Under optimal conditions, the turnaround time for PCR results is as short as 4-6 h. Real-time PCR is a rapid and inexpensive approach that can be easily introduced into clinical practice for detection of organisms directly from tissue samples. Characterization of the anaerobic microflora by real-time PCR of chronic wounds is warranted.
authors.affiliation: Infectious Diseases, Johns Hopkins Medical Institutions, Johns Hopkins Wound Center at Bayview, 5501 Hopkins Bayview Circle, Baltimore, MD 21224, USA. jmelend3@jhmi.edu
authors.forename: J H
authors.initials: JH
authors.lastname: Melendez
authors.forename: Y M
authors.initials: YM
authors.lastname: Frankel
authors.forename: A T
authors.initials: AT
authors.lastname: An
authors.forename: L
authors.initials: L
authors.lastname: Williams
authors.forename: L B
authors.initials: LB
authors.lastname: Price
authors.forename: N-Y
authors.initials: NY
authors.lastname: Wang
authors.forename: G S
authors.initials: GS
authors.lastname: Lazarus
authors.forename: J M
authors.initials: JM
authors.lastname: Zenilman
mesh_terms: D001420:Bacteria, Aerobic / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D001424:Bacterial Infections / Q000175:diagnosis* / Q000382:microbiology; D015373:Bacterial Typing Techniques; D015169:Colony Count, Microbial; D003470:Culture Media; D004269:DNA, Bacterial / Q000032:analysis / Q000302:isolation & purification; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors; D014947:Wounds and Injuries / Q000382:microbiology*
chemical_list: D003470:Culture Media; D004269:DNA, Bacterial