Fractuur-gerelateerde infectie: optimalisatie van diagnostische principes en de ontwikkeling van alternatieve strategieën voor preventie en behandeling

Fracture-related infection (FRI) remains an important problem after musculoskeletal trauma surgery. At the center of these infections are microorganisms that form biofilms on the surface of implanted devices and necrotic bone. These biofilms shield the bacteria from the host immune system and are tolerant to most antibiotics, even in the absence of specific antibiotic resistance genes. All biofilm-related infections are, therefore, considered to be essentially antibiotic-resistant and a challenge to treat. Although extensive studies and guidelines have been published for its orthopedic counterpart - periprosthetic joint infection (PJI) - scientific research on FRI has lagged behind. For the above-mentioned reasons, there is an important clinical need for new insights in the field of FRI, whereby early diagnosis and the development of new prevention strategies have become increasingly important. Furthermore, in times when antimicrobial resistance is a worldwide problem, the development of alternative treatment strategies assumes greater importance. In the first part of this thesis, diagnostic principles for FRI were investigated. In Chapter 1, a systematic review is described that summarizes the evidence for the use of sonication fluid culture tests, histopathology and molecular diagnostics. Ten studies were included of which five focused on sonication fluid culture, three on histopathology and two on molecular diagnostics. Regarding sonication, although all five studies showed encouraging results, the evidence is of low quality and we could not conclude that sonication fluid culture was superior to standard deep tissue cultures. It could, however, be a useful adjunct to standard tissue cultures, especially in patients who have received antibiotic treatment preoperatively. Regarding histopathology and molecular techniques, evidence was even more scarce and based on small studies. The study in Chapter 2 provides a retrospective evaluation of diagnosing long bone FRI, comparing tissue cultures retrieved from the Reamer-Irrigator-Aspirator (RIA) system to standard deep tissue cultures. The RIA-system is used for reaming of the intramedullary canal, which is critical to remove debris surrounding the removed device (i.e., intramedullary nail) and within the canal. Bone overheating and bacterial seeding - which are serious concerns with standard intramedullary reaming - are minimized due to the use of a disposable sharp reaming head, cold irrigation and aspiration of the debris in a filter. As a proof-of-concept, this cohort study showed that the RIA-system could have a potential role in the diagnosis of FRI as an adjunct to standard tissue cultures, especially in cases where it is difficult to retrieve adequate and a sufficient amount of tissue cultures. However, further research on this topic is required before its routine application in clinical practice. In Chapter 3, the consensus definition for FRI was validated in a multicentric, retrospective study. Patients who underwent revision surgery due to a suspicion of FRI were included in the study. We used 'intention to treat' recommended by the multidisciplinary team as a reference standard to subdivide patients into the FRI group or the control group. Our results show that the presence of any of the confirmatory diagnostic criteria excluding histopathology are associated with a sensitivity of 97.5%, a specificity of 100% and a near perfect diagnostic discriminatory value of 0.99 for FRI. Adequate tissue sampling should be regarded as an indispensable step in diagnostic decision-making and towards appropriate treatment. Our results suggest that, when a single deep tissue culture is positive with a virulent pathogen, this should raise suspicion that an FRI may be present. Regarding radiological signs, our results confirm that radiological evaluation will add little information regarding the presence or absence of FRI. Further research is required regarding the diagnostic value of nuclear imaging tests. Although WBC count had a high specificity in our study, it did not increase the discriminatory value when evaluated with other confirmatory and suggestive clinical signs. Serum inflammatory markers should therefore remain suggestive signs for infection. They may be more useful to monitor the patient during the follow-up period. On the other hand, the presence of clinical suggestive signs such as local warmth/swelling/redness, wound drainage and fever should raise suspicion that an FRI may be present. However, when these parameters are not present, FRI cannot be ruled out. These results underscore the importance of carefully evaluating the presence of sets of confirmatory and suggestive diagnostic criteria, instead of relying on a single criterion. The second part of this thesis focused on alternative prevention and treatment strategies for FRI. The narrative review in Chapter 4 provides an overview of the state-of-the-art in phage therapy for orthopedic device-related infections (ODRIs). Not many in vitro phage studies have been performed using ODRI-associated strains, but many studies have proven the ability of phages to inhibit biofilm formation. Furthermore, the available in vivo studies show a high efficacy concerning the eradication of the infection, but the methodology is not comparable between studies. Clinical studies have shown promising results in patients with severe musculoskeletal infections, including ODRIs. Most of these studies have shown the added value of concomitant antibiotics. Important to note is that the earlier clinical trials conducted in Eastern Europe did not conform to the good clinical practice guidelines known today. More recently, sporadic phage applications have been performed in Western Europe under the umbrella of the Article 37 of the Helsinki Declaration. Even though no safety issues were reported and most targeted infections seemed to have been resolved, the small number and diversity of these 'Helsinki' phage therapy cases so far did not allow for the unambiguous demonstration that the positive clinical outcome was related to phage use. Also, there remains a lack of data concerning phage production, processing, administration and dosing, as well as follow-up clinical monitoring reports. To fully implement phage therapy in the treatment of ODRI, an integrated clinical approach is required, supported by comprehensive legislature to enable expansive and correctly implemented clinical trials. In this regard, the first steps towards the establishment of a 'multidisciplinary phage task force' (MPTF) and a standardized treatment pathway are presented in Chapter 5, based on our experience of four patients with severe musculoskeletal infections. Based on the isolated pathogens, phage cocktails were selected and applied intraoperatively. A draining system allowed concomitant postoperative administration for a seven to ten days, three times per day. All patients received concomitant antibiotics and their clinical status was followed daily during phage therapy. No severe side-effects related to the phage application protocol were noted. None of the patients developed neutralizing antibodies against the applied phages. After a single course of phage therapy with concomitant antibiotics, no recurrence of infection with the causative strains occurred, with follow-up periods currently ranging from 28 to 36 months. This approach was continued after the publication of this study. Moreover, Chapter 6 presents a study protocol for a prospective, observational registry study regarding the application of phage therapy in three distinct medicinal disciplines: musculoskeletal infection, chronic rhinosinusitis and sepsis. This study aims at collecting safety and efficacy data of patients who were treated with phage therapy. The previously mentioned MPTF, from now on referred to as the Coordination group for Bacteriophage therapy Leuven (CBL), plays a central role in patient selection, the setup of the treatment plan and the follow-up of the patient. The aim of the study described in Chapter 7 was to provide an in vivo proof-of-concept regarding the prevention and treatment of FRI by applying bacteriophages (either in suspension in saline or in a phage-loaded hydrogel). A single application of phage in saline was highly effective in preventing the infection. In a treatment setting, daily administration of phage in saline through a port system was compared to a single application of a phage-loaded hydrogel and a control group receiving systemic antibiotics only. A possible trend of bacterial load reduction on the implant was observed with the phage-loaded hydrogel. However, phage therapy did not result in a statistically significant reduction of the bacterial load. The application of phage in saline through a port system was complicated by superinfection and the development of neutralizing antibodies. The latter was not found in the animals that received the phage-loaded hydrogel, which may indicate that encapsulation of phages into a carrier such as a hydrogel limits their exposure to the adaptive immune system. These studies showed that phage therapy can be useful in targeting orthopedic device-related infections, however, further research and improvements of the application methods are required. The concept of local delivery systems has gained interest. Currently, most fracture fixation devices are made out of metals such as stainless steel or titanium alloy, but they lack a biologically active surface that can prevent biofilm formation or encourage osteointegration. The study in Chapter 8 evaluated the biocompatibility of a 2-aminoimidazole (2-AI) based antibiofilm coating. In contrast to classic antimicrobials the 2-AI alkaloids specifically target biofilm formation, without affecting the free-living growth of bacteria. This renders bacteria more susceptible to antibiotic treatment. The 2-AI based compound was covalently linked to titanium orthopedic implants and evaluated in a clinically relevant rabbit fracture model. The activity of the coating was confirmed in vitro. An in vivo biocompatibility and healing study indicated that the coated implants did not negatively affect fracture healing or osteointegration. The next step will be to validate the coating in an infection setting.

Publication year:2021

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