Culture of multiple intraoperative tissue samples is the standard of microbiological diagnosis of prosthetic joint infections. Recently, improved sensitivity of using prosthesis sonication method and molecular techniques has been reported in the literature. However, collecting the removed prosthesis as well as additional specimens for molecular analysis is not straightforward for the surgeons and assistants in the operation theatre. Our All-in-a-Box concept addresses the need for simple and unambiguous sampling of clinical specimens in the operating theatre, and to overcome the variation in sampling technique within and between surgical teams and across different hospitals.

The All-in-a-Box concept was developed in close cooperation between surgeons, their operating assistants, clinical microbiologists and molecular biologists in order to ensure the concept is easily implemented in the operating theatre, achieving high completeness, and being well preserved all the way to the laboratory.

All needed equipment, vials and forms are collected in a single box, and corresponding items are clearly color coded to further reduce the likelihood of confusion.

Boxes are designed to address the specific needs for either routine diagnosis or special demands as in clinical studies. Their design is based on large experience in connection with diagnosis of joint prosthesis-related infections. Downstream SOPs for sample processing are included in the All-in-a-box concept and specimens can subsequently be analyzed in parallel by culturing and molecular methods.

We have implemented this concept in two large research projects, we received 1508 (89%) of 1685 scheduled samples during the 2-year project period in the first project despite several different surgical teams and hospitals, while the other project is still ongoing.

All-in-a-Box is useful concept to improve the completeness of routine sampling for microbial analysis.

Identification of modalities and procedures to improve the differential diagnosis of septic and aseptic cases in patients with joint-related pain after total hip or knee alloplasty (THA/TKA).

A prospective cohort of 147 patients presenting with problems related to previous THA or TKA was included and subjected to a comprehensive diagnostic algorithm. The standard diagnostics were supplemented with novel or improved methods for sampling of clinical specimens, sonication of retrieved implant parts, prolonged and effective culture of microorganisms, and dedicated clinical samples for molecular biological detection and identification of microorganisms. Furthermore, comprehensive pain investigations and nuclear imaging were employed. For each case the clinical management was decided upon in a clinical conference with participation of clinical microbiologist, orthopedics and experts in nuclear imaging. The clinical management of patients was blinded against the molecular biological detection of microorganisms.

Patients grouped as follows: 69 aseptic, 19 acute septic, 19 chronic septic, 40 pain/unresolved. Sonication of retrieved implant parts resulted in detection of biofilm not detected by standard specimens, i.e. joint fluid and periprosthetic tissue biopsies. Next generation sequencing detected and identified few infections not detected by culture. Molecular analyses showed more polymicrobial infections than culture. Nuclear imaging was inconclusive with respect to recommendation of changed setup. Analysis of blood based biomarkers is ongoing. Patients with chronic pain are undergoing follow-up.

The special emphasis put on detection of infections resulted in detection of infections in joints that otherwise would have been categorized as aseptic loosening. Clinical management for these cases was changed accordingly. The cross-disciplinary clinical conference is considered valuable for clinical management. The clinical relevance of the polymicrobial nature of infections as diagnosed employing next generation sequencing is yet to be established. Long-term follow-up is planned.

Prosthetic joint infection is one of the most challenging complications of joint alloplasty and the diagnosis remains difficult. The aim of the study was to investigate the bacterial flora in surgical samples from 22 prosthetic patients using a panel of culture-independent molecular methods including broad range 16S rRNA gene PCR, cloning, sequencing, phylogeny, quantitative PCR (qPCR), and fluorescence in situ hybridization (FISH). Concomitant samples were cultured by standard methods.

Molecular methods detected presence of bacteria in samples from 12 of 22 patients. Using clone libraries a total of 40 different bacterial species were identified including known pathogens and species not previously described in association with prosthetic joint infections. The predominant species were Propionibacterium acnes and Staphylococcus epidermidis; polymicrobial infections were found in 9 patients. Culture-based methods showed bacterial growth in 8 cases with the predominant species being S. epidermidis. Neither anaerobic bacteria (including P. acnes) nor any of the species not previously described in implant infections were isolated. Additionally, 7 of the 8 culture positive cases were monomicrobial. Overall, the results of culture-based and molecular methods showed concordance in 11 cases (hereof 9 negative by both methods) and discrepancy in 6 cases. In the remaining 5 cases, culture-based methods identified only one species or a group of bacteria (e.g., coagulase negative staphylococci or coryneform rods), while culture-independent molecular methods were able to detect several distinct bacterial species including a species within the group identified by culture. A qPCR assay was developed to assess the abundance of Propionibacterium while S. aureus was quantified by a published S. aureus qPCR assay. These quantifications confirmed the findings from the clone library approach and showed the potential of qPCR for fast detection of bacteria in orthopedic samples. Additionally, both single cells and microcolonies were visualized using FISH and confocal scanning laser microscopy.

In conclusion, the molecular methods detected a more diverse bacterial flora in prosthetic joint infections than revealed by standard culture-based methods, and polymicrobial infections were more frequently observed. The pathogenesis of these microorganisms and their role in implant-associated infections needs to be determined.