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.
1Department of Orthopaedic Surgery, George Washington University, 2150 Pennsylvania Avenue N.W., Washington, DC 20037
2Anderson Orthopaedic Research Institute, 2501 Parker's Lane, Alexandria, VA 22306
3Cartilage 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
4Department 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: email@example.com