PMA concentration, dark incubation, and photo-activation time periods were optimised. Each one of these parameters was tested separately while the other two variables were kept constant. As a first stage, these conditions were tested on laboratory-prepared live and dead bacterial suspensions. In a second stage, to mimic a clinical situation, the optimised conditions were tested using artificially spiked human tissue homogenates for validation. Organisms used in the optimisation were isolated from PJI cases in our laboratory.
Suspensions of Staphylococcus aureus, being the commonest PJI pathogen, were prepared with a concentration of 105 colony-forming units (cfu)/mL in phosphate buffered saline (PBS) and divided into halves. One half was killed by heating in a dry block at 95°C for 10 min while the other half was left viable. Bacterial killing was defined as a lack of growth after incubation on blood agar for 48 h.
Each of the live and dead portions was further divided into two halves; one of each was treated with PMA (Biotium, Fremont, USA) and the other half left untreated. Dark incubation was done at 37 °C in a shaking incubator for adequate mixing. The PMA-Lite™ LED Photolysis Device (Biotium) was used for the photo-activation of the PMA.
GenuElute DNA extraction kit (Sigma-Aldrich, Missouri, USA) was used for bacterial DNA extraction following the manufacturer’s protocol. PCR was run using Mx3005P QPCR (Agilent, California, USA). Each sample was run in duplicate in the same PCR assay and each test was repeated three times. For each of the live and dead samples, difference in cycle threshold Ct (ΔCt) was calculated, where: ΔCt viable is the difference between the Ct value of viable bacteria with and without PMA treatment and ΔCt dead is the difference between the Ct value of dead bacteria with and without PMA treatment. The ideal PMA treatment conditions should have the highest ΔCt dead and lowest ΔCt viable.
For PMA concentration optimisation, four different PMA concentrations were tested: 10, 20, 50 and 100 μM. After addition of the PMA, samples were incubated in the dark for 5 min and then photo-activated for 20 min.
For optimisation of dark incubation, a concentration of 20 μM was used. 5, 10, and 30 min were tested with the same photo-activation time of 20 min.
For photo-activation optimisation, a concentration of 20 μM and a five-minute dark incubation were used. 10, 20, and 30 min were tested.
Application of optimised PMA in clinical samples
Using the optimum conditions above, clinical samples were examined. 202 periprosthetic tissues and/or explanted prosthesis samples were collected from 60 episodes in 58 patients undergoing revision arthroplasties due to either PJI or non-infective causes (eg aseptic loosening or periprosthetic fracture). The Musculoskeletal Infection Society (MSIS) definition of PJI was used . Figure 1 is an overview of the processing of clinical samples.
Tissues were homogenized using the Roche magNA Lyser homogenizer (Hoffman-La Roche Ltd, Basel Switzerland) at a speed of 4500 rpm for four cycles, each lasting 45 s . Prostheses were sonicated in a precision sonicator set at 50 Hz (Ultrawave Ltd, Cardiff, UK) for 5 min.
Homogenates and sonicates were used for aerobic and anaerobic cultures, PCR (PCR without PMA), and PMA-PCR (viability PCR). For PMA treatment, 5 µL of 2 mM of the photo-reactive dye was added to the 500 mL sample in a dim-light room for a final concentration of 20 μM/µL. The sample was then incubated in the dark for 5 min (covered with aluminium foil), while gently shaking the tubes to ensure proper mixing. PMA-Lite™ LED Photolysis Device was used for the photo-activation for 10 min.
For DNA extraction, the GenuElute DNA extraction kit was used according to the manufacturer’s instructions.
The PCR assay included genus-specific primers for staphylococci  and enterococci  and species-specific primers for Cutibacterium acnes . The human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeping gene  was amplified as an internal control of DNA extraction and possible PCR inhibition (Table 1). Separate PCR assays were used for each primer pair.
A final volume of 20 μL was prepared in each tube by mixing 10 μL of SensiFAST™ SYBR® Lo-ROX Master Mix Kit (Bioline Reagents Ltd, London, UK), 0.5 μL of each of the forward and reverse primer, 4 μL nuclease free water and 5 μL template or nuclease free water for non-template control (NTC) preparation. NTC samples were used both in PMA-treated and non-treated samples to confirm non - contamination of the PMA. The terms viability PCR and conventional PCR are used in this manuscript to refer to PCR with and without PMA treatment respectively.
A positive tissue culture was defined as the isolation of the same organism from two or more tissue samples. A cutoff value of 101 cfu/mL for the sonicate culture positivity was used as recommended by Trampuz et al. . Collectively, a positive culture per episode was defined as either two or more positive tissue samples and/or positive sonicate culture.
A positive PCR sample was defined as any significant amplification (using the dissociation curve and/or gel electrophoresis) with a Ct value above the detection threshold. Definition of a positive PCR episode is an episode with two or more positive tissue samples and/or positive sonicate in the same assay.
Effect of PMA on sensitivity of PCR assay in clinical samples
Ten-fold serial dilution suspensions of fresh culture (18–24 h old) of E. faecalis were prepared from 108 to 101 cfu/mL. Non-infected tissue homogenates were tested with PCR to ensure they were E. faecalis-free. 500 mL of each bacterial suspension concentration was added to the same amount of tissue homogenate and then divided; one half was treated with PMA and the other left untreated. DNA was then extracted and PCR run as described above. Samples were run in duplicate in each experiment and three independent experiments were carried out on three different days. In addition to a positive control and NTC, a clean tissue sample was tested to further ensure lack of contamination. Ct values of PMA treated samples were compared to their untreated equivalents.
Graphpad Prism 7 was used to analyse data and produce charts. Appropriate statistical tests were used according to the distribution of data. p values < 0.05 were considered significant. Using the above mentioned definitions, sensitivities, specificities, and accuracies of culture, PCR, PMA-PCR were calculated for the episodes rather than samples.
Human tissue samples were collected under the ethics approval of the Nottingham Health Science Biobank.