0
Scientific Articles   |    
Detection of Periprosthetic Infections With Use of Ribosomal RNA-Based Polymerase Chain Reaction
Patrick F. Bergin, MD1; Jason D. Doppelt, MD1; William G. Hamilton, MD2; Gudrun E. Mirick, MD1; Angela E. Jones, MD1; Supatra Sritulanondha, MPH2; Jeannine M. Helm, MS3; Rocky S. Tuan, PhD4
1 Department of Orthopaedic Surgery, George Washington University, 2150 Pennsylvania Avenue N.W., Washington, DC 20037
2 Anderson Orthopaedic Research Institute, 2501 Parker's Lane, Alexandria, VA 22306
3 Cartilage Biology and Orthopaedics Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Department of Health and Human Services, Room 1140, Building 50, MSC 8022, Bethesda, MD 20892-8022
4 Department of Orthopaedic Surgery, Center for Cellular and Molecular Engineering, University of Pittsburgh School of Medicine, 450 Technology Drive, Room 221, Pittsburgh, PA 15219. E-mail address for R.S. Tuan: rst13@pitt.edu
View Disclosures and Other Information
Disclosure: The authors did not receive any outside funding or grants in support of their research for or preparation of this work. One or more of the authors, or a member of his or her immediate family, received, in any one year, payments or other benefits or a commitment or agreement to provide such benefits from commercial entities in excess of $10,000 (DePuy, a Johnson and Johnson company) and less than $10,000 (Lifenet). This research was supported in part by the National Institute of Arthritis and Musculoskeletal and Skin Diseases Intramural Research Program (NIH ZO1 AR41131).

Investigation performed at George Washington University, Washington, DC; National Institutes of Health, Bethesda, Maryland; Anderson Orthopaedic Research Institute, Alexandria, Virginia; and University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania

Copyright ©2010 American Society for Journal of Bone and Joint Surgery, Inc.
J Bone Joint Surg Am, 2010 Mar 01;92(3):654-663. doi: 10.2106/JBJS.I.00400
5 Recommendations (Recommend) | 3 Comments | Saved by 3 Users Save Case

Abstract

Background: 

Previously described molecular biology techniques used to detect periprosthetic infections have been complicated by false-positive results. We have reported the development of a messenger RNA (mRNA)-based procedure to reduce these false-positive results. The limitations of this procedure are the lack of a universal target and reduced sensitivity due to a low concentration of bacterial mRNAs in test samples. The objective of the present study was to determine whether reverse transcription-quantitative polymerase chain reaction (RT-qPCR) using universal primers can be used to detect the more abundant bacterial ribosomal RNA (rRNA) as an indicator of periprosthetic infection.

Methods: 

Serial dilutions of simulated synovial fluid infections were analyzed with rRNA RT-qPCR to determine the detection limit of this assay. Escherichia coli cultures treated with gentamicin were analyzed with RT-qPCR over a twenty-day time course to determine the degradation of rRNA as compared with the decrease in the viable cell count as determined by means of cell plating. As a proof of concept, group-specific polymerase chain reaction primers were developed for Streptococcus species and were tested against fifteen orthopaedically relevant organisms to show the potential for speciation with this assay. Sixty-four patients with a symptomatic effusion at the site of a total knee arthroplasty were enrolled, and complete patient information was documented in a prospective manner. Synovial fluid analysis with rRNA RT-qPCR was performed in a blind fashion.

Results: 

The rRNA RT-qPCR assay was able to detect as few as 590 colony forming units/mL of Staphylococcus aureus and 2900 colony forming units/mL of Escherichia coli. The rRNA RT-qPCR signal closely followed cell death, pointing to its potential use as a viability marker. Three group-specific primer sets correctly identified their intended targets without amplifying closely related species. Clinically, the test correctly identified all six patients with a confirmed infection and all fifty patients who clearly did not have an infection. Eight patients had some laboratory or clinical signs of infection, but their status could not be confirmed. Infection was indicated by rRNA RT-qPCR in three of these patients who had elevated synovial fluid white blood-cell counts but negative results on culture. For statistical purposes, all patients who were categorized as indeterminate were considered to have an infection for the purpose of analysis, for a prevalence of 22% in this cohort.

Conclusions: 

With respect to current diagnostic tests, rRNA-based RT-qPCR demonstrated 100% specificity and positive predictive value with a sensitivity equivalent to that of intraoperative culture. The RT-qPCR signal followed bacterial culture trends but exhibited detectable level for seven days after sterilization, allowing for the detection of infection after the antibiotic administration. These findings indicate that rRNA RT-qPCR is a sensitive and reliable test that retains the universal detection and speciation of DNA-based methods while functioning as a viability indicator.

Level of Evidence: 

Diagnostic Level I. See Instructions to Authors for a complete description of levels of evidence.

Figures in this Article
    Sign In to Your Personal ProfileSign In To Access Full Content
    Not a Subscriber?
    Get online access for 30 days for $35
    New to JBJS?
    Sign up for a full subscription to both the print and online editions
    Register for a FREE limited account to get full access to all CME activities, to comment on public articles, or to sign up for alerts.
    Register for a FREE limited account to get full access to all CME activities
    Have a subscription to the print edition?
    Current subscribers to The Journal of Bone & Joint Surgery in either the print or quarterly DVD formats receive free online access to JBJS.org.
    Forgot your password?
    Enter your username and email address. We'll send you a reminder to the email address on record.

     
    Forgot your username or need assistance? Please contact customer service at subs@jbjs.org. If your access is provided
    by your institution, please contact you librarian or administrator for username and password information. Institutional
    administrators, to reset your institution's master username or password, please contact subs@jbjs.org

    References

    Accreditation Statement
    These activities have been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Academy of Orthopaedic Surgeons and The Journal of Bone and Joint Surgery, Inc. The American Academy of Orthopaedic Surgeons is accredited by the ACCME to provide continuing medical education for physicians.
    CME Activities Associated with This Article
    Submit a Comment
    Please read the other comments before you post yours. Contributors must reveal any conflict of interest.
    Comments are moderated and will appear on the site at the discretion of JBJS editorial staff.

    * = Required Field
    (if multiple authors, separate names by comma)
    Example: John Doe





    Related Content
    The Journal of Bone & Joint Surgery
    JBJS Case Connector
    Topic Collections
    Related Audio and Videos
    PubMed Articles
    Clinical Trials
    Readers of This Also Read...
    JBJS Jobs
    02/10/2014
    IL - The University of Chicago's Department of Orthopaedic Surgery and Rehabilitation Medicine
    03/19/2014
    VA - OrthoVirginia
    06/29/2012
    PA - Thomas Jefferson University