Reliability of microbiological diagnosis of prosthetic joint infection [PJI] strongly depends on the ability to dislodge microorganisms from biofilm and on the rate of contaminating samples during collection in the operating room and processing. The aim of a correct protocol is to avoid false negative and false positive results in order to adapt the correct therapy for each patient.

The object of the present study was to evaluate the impact of a novel closed bag system designed for samples collection and processing based on dithiothreitol (DTT), which is a sulfydryl compound able to remove bacteria from biofilm (MicroDTTect, 4i, Italy), on isolation of contaminant microorganisms in hip prostheses.

Specimens (prostheses, spacers, periprosthetic tissues) were aseptically collected according to a standard protocol into the device, which was transported to the laboratory for culture. Three different models of the system were prospectively evaluated, each being a development of the previous one. The first generation device consisted in an “open” system (DTT eluate was collected with a syringe and dispensed into sterile tubes), the second generation device in a “partially closed” system (DTT eluate collected directly in sterile vacuum tubes) and the third generation device in a “completely closed system” (DTT reservoir directly connected with sealed tubes inside the device). PJI was diagnosed following criteria established by MSIS.

The overall contamination rate, sensitivity and specificity of the first generation “open” system MicroDTTect were respectively 2.6% (1/39), 82.3% and 95.4% in 39 hips. The second generation “partially closed” device was characterized by a contamination rate of 1.96% (1/51), a sensitivity of 84% and a specificity of 96.1% in 51 hips. Contamination rate further decreased in the third generation “closed” system (1.89%, 2/106), while sensitivity (91.3%) and specificity (96.7%) improved in 106 hips. Differences have been also observed in hips (106) when compared to knees (70 cases) prosthetic infections (sensitivity 91.3% vs 89.3% and specificity 96.7% vs 100%).

Our data show as, thanks to its ease of use, low contamination rate and high sensitivity, MicroDTTect can represent a useful tool for improving the microbiological diagnosis of PJIs in hip revisions and has replaced sonication in our practice.

Aim

Biofilm-related infections represent a recurrent problem in the orthopaedic setting. In recent years, great interest was directed towards the identification of novel molecules capable to interfere with pathogens adhesion and biofilm formation on implant surfaces. In this study, two stable forms of α-tocopherol, the hydrophobic acetate ester and the water-soluble phosphate ester, were tested in vitro as coating for titanium prostheses.

Development of antibacterial surfaces or coatings to prevent bacterial adhesion and hence colonization of implants and biofilm formation is an attractive option, in order to reduce the tremendous impact of implant-related infections associated with modern surgery. To overcome the lack of in vivo and clinical models, able to evaluate the performance of anti-adhesive coatings, we designed an in vitro experimental setting that allows to quantitatively evaluate the ability of a coating to reduce bacterial adhesion on a given surface; this model may efficiently serve as a surrogate endpoint to validate anti-adhesive medical devices and compounds. Here we report the results the evaluation of the anti-adhesive properties of a patented, fast-resorbable hydrogel coating, (“Defensive Antibacterial Coating”, DAC).

Sterile sandblasted titanium discs of approximately 5cm² surface area were used as substrates for bacterial adhesion. The gel was prepared as follows: syringes prefilled with 300 mg of DAC powder (Novagenit Srl) were reconstituted with 5 ml of sterile water to obtain a hydrogel with a DAC concentration of 6%. Two experiments were conducted. In the first, 200 mg of hydrogel were homogenously spread on the surface of titanium disc, with the spreading device provided by the manufacturer. Both coated and uncoated substrates (controls) were overlaid with a standardized inoculum (10⁸ CFU/ml) of a wild methicillin-resistant Staphylococcus aureus strain, previously isolated from a peri-prosthetic joint infection, for 15, 30, 60 and 120 minutes. Afterwards, non-adherent bacteria were removed by rinsing with sterile saline. The remaining adhered cells were seeded on agar plates for CFU count. In the second experiment, the discs were first inoculated with bacterial cells followed by a treatment with the hydrogel and bacterial count as described above. Ten discs were used for each condition and each time interval (total 160 discs).

The adhesion density of S. aureus on titanium discs pre-treated with DAC was significantly lower than that observed on untreated controls at each time point. In particular, the average number of adherent bacteria at 15, 30, 60 and 120 minutes of incubation, was respectively reduced by 86.8%, 80.4%, 74.6% and 66.7%, compared to controls (p<0.001). DAC treatment of discs with previously adhered S. aureus reduced bacterial adhesion, at 15, 30, 60 and 120 minutes of incubation, by, respectively, 84.0% (p<0.05), 72.8%, 72.3% and 64.3% (p<0.001), compared to untreated controls.

Our results shows that DAC, “Defensive Antibacterial Coating”, has anti-adhesive properties that allow to reduce bacterial adhesion on a sanded titanium surface by more than 80%, even in the presence of remarkably high bacterial loads (10⁸ CFU/ml), of multi-resistant bacteria (MRSA) and even in the case of previous contamination. Providing anti-adhesive properties to a surface with a fast-resorbable coating may be a safe option to protect inorganic and organic surfaces and biomaterials. Those observation could be the pre-requisite for its in vivo application.

Prosthetic implants, periprosthetic and osteoarticular tissues are specimens of choice for diagnosis of bone and joint infections including prosthetic joint infections (PJIs). However, it is widely known that cultures from prostheses and tissues may fail to yield microbial growth in up to one third of patients. In the recent past, treatment of prosthetic implants have been optimized in order to improve sensitivity of microbiological cultures, while less attention has been addressed to tissue samples. For these latter homogenization is considered the best procedure, but it is quite laborious, time-consuming and it is not always performed in all laboratories. Dithiothreitol (DTT) has been proposed as an alternative treatment to sonication for microbiological diagnosis of PJIs. In this study, we evaluated the applicability of MicroDTTect treatment, a closed system developed for transport and treatment of tissues and prosthetic implants with DTT.

For evaluation of applicability of MicroDTTect to tissue specimens, samples (tissues and, in case of PJI, prosthetic implants) from 40 patients (12 PJIs and 5 osteomyelitis and 23 not-infected) were evaluated. MicroDTTect system consists of a sterile plastic bag containing a reservoir which allows for release of a 0.1% (v:v) DTT solution, once the sample is placed into the bag. Comparison of MicroDTTect treatment of prostheses with sonication included samples from 30 patients (14 with aseptic loosening of the prosthesis and 16 with PJIs). Of two tissue samples from the same region, one was placed into MicroDTTect bag and the other was collected in a sterile container with addition of sterile saline. After agitation and centrifugation of the eluate, aliquots of the pellets were plated on agar plates and inoculated into broths which were incubated for 48 hrs and 15 days, respectively.

Treatment of prosthetic implants with MicroDTTect showed a higher specificity and sensitivity than sonication (specificity 92.8% vs 85.7%; sensitivity: 87.5% vs 75.0 % DTT vs sonication). When used for tissue treatment, MicroDTTect showed a sensitivity of 82.3% and a specificity of 97% which were higher than that observed when saline was used (sensitivity: 64.7%; specificity 91%).

Treatment of tissues and prosthetic implants with MicroDTTect may be a practicable strategy to improve microbiological diagnosis of osteoarticular infections, reducing sample manipulation and therefore limiting sample contamination. Moreover, use of MicroDTTect does not require dedicated instrumentation, and is time and cost saving.