Detection of periprosthetic infections with use of ribosomal RNA-based polymerase chain reaction

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.

Fig. 1

Degradation of mRNA and rRNA with cell death. Cell cultures of Escherichia coli (1.4 × 10⁹ CFU/mL) were treated at Day 0 with 1 mg/mL gentamicin. Over a twenty-day period, the concentration of cells (CFU/mL) was calculated by means of a traditional agar plating technique (blue line). The cells were pelleted by means of centrifugation and were resuspended in clean growth media three times to ensure that there was no remaining antibiotic (which would have interfered with the plating results). In addition, at each time point, total RNA extraction was performed and cell concentration was predicted with use of mRNA RT-qPCR (green line) and rRNA RT-qPCR (red line). The absence of viable bacteria is predicted by RT-qPCR when the calculated cell concentration reaches the detection threshold indicated by the dashed lines (6.0 × 10⁴ for mRNA and 2.9 × 10³ for rRNA). (For the rRNA data, although a theoretical limit of 2.9 x 10² CFU/mL is indicated based on theoretical extrapolation, the experimental data could only reliably detect 2.9 x 10³ CFU/mL, while the difference between 2.9 x 10² and zero could not be accurately ascertained.)

Fig. 2

Species-specific rRNA-based RT-qPCR. rRNA RT-qPCR analysis of RNA samples was performed on total RNA obtained from fifteen clinically relevant bacterial pathogens with use of group-specific primers. All organisms were grown to a concentration of 1 × 10⁷ CFU/mL prior to total RNA extraction. The three graphs show the polymerase chain reaction outcome with use of primers designed for (a) group-A Streptococcus, (b) group-B Streptococcus, and (c) α-hemolytic Streptococcus. Initiation of the logarithmic rise in the analysis curve at a lower cycle indicates identification of the labeled species. Arrows indicate resultant Ct curves from polymerase chain reactions with use of RNA from the intended target organisms. All reactions were run with duplicate samples.