Simulated Joint Infection Assessment by Rapid Detection of Live Bacteria with Real-Time Reverse Transcription Polymerase Chain Reaction

Abstract

The direct quantification of the concentration of viable bacterial mRNA with real-time quantitative reverse transcription polymerase chain reaction allows identification of both culture-positive bacterial infection and so-called unculturable bacterial infection in a simulated septic arthritis model. In contrast to conventional polymerase chain reaction, real-time quantitative reverse transcription polymerase chain reaction minimizes false-positive detection of nonviable bacteria and thus provides relevant information on the success or failure of antibiotic therapy.

Detection of Periprosthetic Infections With Use of Ribosomal RNA-Based Polymerase Chain Reaction

“…Thus, in principle, an RNA-based polymerase chain reaction method would be preferable as RNAs are more labile and would be associated primarily with viable cells. Our recent study involving mRNA-based RT-qPCR illustrates the feasibility of this approach 17 , albeit with an expected reduced sensitivity in comparison with the DNA-based methods.…”

“…Total RNA extraction was performed as described above on each sample in the dilution series for subsequent RT-qPCR analysis with universal rRNA primers as described below. The same total RNA sample was also analyzed with mRNAbased RT-qPCR with use of previously described primers 17 .…”

“…For both species, there was a linear relationship between logarithmic bacterial concentration and DCt. The same total RNA samples were also analyzed by means of quantitative mRNAbased RT-qPCR 17 . The latter method yielded a detection limit of 60,000 CFU/mL for Escherichia coli and 110,000 CFU/mL for Staphylococcus aureus.…”

“…With the persistence of bacterial DNA after cell death, any bacterial contamination, even in an antibiotic-cleared infection, could cause a positive result in an otherwise sterile sample. In our recent study, we explored the utility of messenger ribonucleic acid (mRNA) as a viability indicator with use of reverse transcription-quantitative polymerase chain reaction (RTqPCR) 17 . In that study of simulated joint infections, there were no false-positive results and all detectable signals quickly approached the sterile baseline.…”

Revision Total Knee Arthroplasty Infection: Incidence and Predictors

“…The latter again highlights the limitations of current diagnostic modality for PJI that fails to detect an organism in some cases. With improvements in diagnosis of periprosthetic infection such as sonication [39] or the use of molecular techniques [3], we believe more of these patients with ''aseptic'' failures will be correctly diagnosed as infected and treated appropriately. In a study by Bongartz et al, the rate of periprosthetic infection was 3.5% in patients with a history of infection in other prosthetic joints [5].…”