Eivind Witsø

Sonication is superior to scraping for retrieval of bacteria in biofilm on titanium and steel surfaces in vitro

Background and purpose Low-virulence implant infections are characterized by bacterial colonization of the implant with subsequent biofilm formation. In these cases, soft tissue biopsies often prove to be culture negative. Consequently, detachment of the causative adherent bacteria is crucial for correct microbiological diagnosis. Using an in vitro model, we compared 4 methods of biofilm sampling from metal surfaces. Methods Discs of titanium and steel were incubated in the presence of Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, and Propionibacterium acnes in Mueller Hinton broth. Non-adherent bacteria were removed by repeated rinsing of the discs. 10 parallels of each disc were subjected to 1 of 4 methods for bacterial recovery: (A) sonication of the discs, (B) scraping of the discs using surgical blades followed by streaking of the blades onto agar plates, (C) scraping of the discs followed by vortex mixing of the surgical blades, and (D) scraping of the discs followed by sonication of the surgical blades. Quantitative bacterial cultures were performed for each sampling method. Results With the exception of S. epidermidis on steel, sonication efficiently and reliably dislodged biofilm bacteria. The scraping methods employed did not detach bacteria embedded in biofilm. Interpretation Scraping of metal surfaces is not an adequate method for sampling of biofilm bacteria in vitro.

Cancellous bone as an antibiotic carrier.

We compared the release characteristics of antibiotics from in vivo and in vitro processed morselized cancellous bone. The bone was impregnated with 7 antibiotics and compressed into a wire-mesh cylinder. In vitro, the bone was processed by daily transfer of the cylinder with its contents into test tubes with broth. The amount of antibiotic eluted from the bone was measured after 1, 3 and 7 days. In vivo, the cylinder was implanted intramuscularly in the interscapular region in rats. After 1, 3 and 7 days, the cylinder was removed and the amount of antibiotic eluted in broth was measured. The results showed that morselized cancellous bone can act as a carrier of antibiotics in vitro and in vivo. The elution profiles of netilmicin-, vancomycin-, clindamycin- and rifampicin-impregnated cancellous bone processed in vitro and in vivo were similar.

Adsorption and release of antibiotics from morselized cancellous bone. In vitro studies of 8 antibiotics.

We studied the basic release patterns of antibiotics from cancellous bone in vitro. Antibiotic-impregnated bone was compressed into a wire-mesh cylinder and the release of antibiotic was assessed by two different in vitro methods: agar diffusion and broth elution. The zones of inhibition were measured on seeded agar and the amounts of antibiotics released in elution tubes were assessed by a bioassay. The study continued for 21 days with daily transfer of the cylinders. The results indicated that benzylpenicillin, dicloxacillin, cephalotin, netilmicin, clindamycin, vancomycin, ciprofloxacin and rifampicin were adsorbed to cancellous bone in vitro. Compared to broth elution, agar diffusion showed a prolonged period of release, owing to the small amounts of antibiotic leaking out of the cylinder into the agar. The betalactams had antibacterial activity in broth for a shorter time than the other antibiotics. The release patterns of the betalactams were similar, in spite of their differences in thermal stability. Only rifampicin showed a concentration higher than MIC for longer than 21 days.

Cortical allograft as a vehicle for antibiotic delivery

Background Infection can be a devastating complication after implantation of a cortical bone allograft. The allograft could act as a vehicle for local antibiotic prophylaxis. Material and methods We studied the release of antibiotics in vitro from cortical bone allografts impregnated with antibiotics for different periods of time. We also studied whether cortical allografts impregnated with antibiotics could eradicate Staphylococcus aureus from an experimentally infected graft in vivo. In the in vitro study, pieces of cortical bone were impregnated with netilmicin, vancomycin, ciprofloxacin and rifampicin for 1 h, 10 h and 100 h. The antibiotics were eluted into phosphate-buffered saline (PBS) for 7 days, with daily transfer of the bone into fresh PBS. In the in vivo study, cortical allografts impregnated with antibiotics were placed in rats intramuscularly. 10 μL of an S. aureus suspension (0.6 × 105 CFU) was placed in the intramedullary cavity. After 15 days, the allografts were removed and examined for bacterial growth. Results The amount of antibiotics released in vitro was influenced by the time used for antibiotic impregnation of the bone. Allografts impregnated with netilmicin, vancomycin and rifampicin effectively eradicated perioperative contamination with S. aureus in vivo. Interpretation This study shows that a cortical bone allograft would be an effective vehicle for local antibiotic delivery.

A comprehensive microbiological evaluation of fifty-four patients undergoing revision surgery due to prosthetic joint loosening.

The diagnosis of a chronic prosthetic joint infection (PJI) is challenging, and no consensus exists regarding how best to define the criteria required for microbiological identification. A general view is that culture of periprosthetic biopsies suffers from inadequate sensitivity. Recently, molecular analyses have been employed in some studies but the specificity of molecular analyses has been questioned, mainly due to contamination issues. In a prospective study of 54 patients undergoing revision surgery due to prosthetic joint loosening, we focused on two aspects of microbiological diagnosis of chronic PJI. First, by collecting diagnostic specimens in a highly standardized manner, we aimed at investigating the adequacy of various specimens by performing quantitative bacteriological culture. Second, we designed and performed real-time 16S rRNA gene PCR analysis with particular emphasis on minimizing the risk of false-positive PCR results. The specimens analysed included synovial fluid, periprosthetic biopsies from the joint capsule and the interface membrane, and specimens from the surface of the explanted prosthesis rendered accessible by scraping and sonication. No antibiotics were given prior to specimen collection. Based on five diagnostic criteria recently suggested, we identified 18 PJIs, all of which fulfilled the criterion of ≥2 positive cultures of periprosthetic specimens. The rate of culture-positive biopsies from the interface membrane was higher compared to specimens from the joint capsule and synovial fluid, and the interface membrane contained a higher bacterial load. Interpretational criteria were applied to differentiate a true-positive PCR from potential bacterial DNA contamination derived from the reagents used for DNA extraction and amplification. The strategy to minimize the risk of false-positive PCR results was successful as only two PCR results were false-positive out of 216 negative periprosthetic specimens. Although the PCR assays themselves were very sensitive, three patients with low bacterial numbers in periprosthetic specimens tested negative by real-time PCR. This overall lowered sensitivity is most likely due to the reduced specimen volume used for PCR analysis compared to culture and may also be due to interference from human DNA present in tissue specimens. According to the protocol in the present study, 16S rRNA gene real-time PCR did not identify more cases of septic prosthetic loosening than did culture of adequate periprosthetic biopsies.

Release of netilmicin and vancomycin from cancellous bone

First, we studied the effect of the following variables used for netilmicin- and vancomycin-impregnation of cancellous bone: a) antibiotic concentration of the impregnation fluid, b) time used for impregnation, c) pH of the impregnation fluid, d) the degree of bone morselizing and e) antibiotic combination. An increase in the antibiotic concentration of the impregnation fluid increased the amount of antibiotics released from bone. In addition, the amount of vancomycin eluted was also dependent on the time used for impregnation. The fraction of the total amount of netilmicin and vancomycin released after 24 h was 80% and 30%, respectively. More netilmicin and vancomycin were eluted from bone impregnated with antibiotics at pH 7 than the amount eluted from bone impregnated at pH 3. More netilmicin was eluted from fine morselized bone than from coarse morselized bone. By combining netilmicin and vancomycin in the impregnation fluid, the release of vancomycin was reduced. Secondly, we analyzed if the release of antibiotics from bone was complete: 99.9% of the total amount of netilmicin adsorbed to the bone was released by elution during 6 weeks. Finally, after implantation of netilmicin-impregnated bone in rabbit femur condyle, we measured netilmicin and vancomycin in serum: peak serum values of netilmicin were 4.2 (3.7-4.7) mg/L 2-3 h postoperatively.

High local concentrations without systemic adverse effects after impaction of netilmicin-impregnated bone

Background When cancellous bone is impregnated with antibiotics the subsequent release of antibiotics from the bone shows a high early release. Hence, impaction of large amounts of netilmicin-impregnated bone may cause toxic netilmicin values in serum.Patients and methods We studied kidney and otovestibular function after impacting 50 g of netilmicin-impregnated cancellous bone during revision hip or knee arthroplasty in 20 patients. The bone was impacted in the acetabulum (n = 8), proximal femur (n = 9) and distal femur/proximal tibia (n = 3). Serum creatinine concentration was measured and audiometry was performed before and after the operation. Netilmicin concentrations in serum, joint fluid, and in urine were recorded postoperatively at regular intervals. We analyzed pharmacokinetics in two study groups receiving bone impregnated with netilmicin (50 mL), at either 50 mg netilmicin/mL (group I) or 100 mg netilmicin/mL (group II).Results Neither netilmicin-induced renal toxicity, nor otovestibular toxicity was registered. Peak serum netilmicin values in group I and group II were 0.9 (0.5–1.3) mg/L and 1.8 (0.6–4.0) mg/L, respectively (p = 0.04). Peak netilmicin concentrations in wound drainage fluid in group I and group II were 237 (9–647) mg/L and 561 (196–1132) mg/L, respectively (p = 0.01). In both groups, netilmicin was recovered in urine samples for approximately 4 weeks.Interpretation 50 grams of cancellous bone impregnated with 100 mg/mL netilmicin solution was impacted in the hip or knee joint with no adverse effects. Extremely high local concentrations of netilmicin in joint fluid were recorded postoperatively. The use of antibioitic-impregnated cancellous could be an option when performing revision of hip and knee prostheses.

Lower limb amputations in Trondheim, Norway

Background and purpose In the city of Trondheim, Norway, diabetic lower-limb amputations accounted for one-third of all lower-limb amputations (LLAs). In an attempt to reduce this rate, a diabetic foot team was established in 1996. We present the incidence of LLA in Trondheim as measured 10 years later. Patients and methods In 2004–07, we registered all LLAs performed in Trondheim and then compared the data with previously published data from 1994–1997. From 1996 through 2006, we registered the activity of the diabetic foot team and we also registered the number of vascular procedures performed on citizens of Trondheim from 1998 through 2006. Results Comparing the two 3-year periods 1994–97 and 2004–07, we observed a decrease in all non-traumatic LLAs. The incidence of diabetic major LLAs per 103 diabetics per year decreased from 4.0 to 2.4, and in patients with peripheral vascular disease we observed a decrease in LLAs from 18 to 12 per 105 inhabitants per year. 5,915 consultations on diabetic subjects were conducted by the diabetic foot team during the period 1996–2006. From 1998 to 2006, the rate of vascular procedures decreased in the non-diabetic population, and was unchanged in diabetic subjects. Interpretation In the population of Trondheim city there appears to have been a reduction in the rate of vascular obstructive lower-limb disease between the two 3-year periods 1994–97 and 2004–07. In our judgment, the decline in diabetic LLA also reflects better care of the diabetic foot.

Improved comfort and function of arm prosthesis after implantation of a Humerus-T-Prosthesis in trans-humeral amputees

The use of arm prosthesis in trans-humeral amputees is limited; due to the cone form of the amputation stump. A Humerus-T-Prosthesis was implanted in three patients to create artificial humerus condyles. Two of the patients were successfully rehabilitated with the application of a new type trans-humeral arm prosthesis. This arm prosthesis had a socket which is suspended and stabilized by the humerus and implant only. Traction and rotational stability were secured by adjustable pressure adaptation around the artificial condyles. The third patient developed a pressure wound over the lateral part of the artificial condyle that later healed. He also was subject to a new trauma with a fracture of the ipsilateral scapula and until now has had limited the use of his new arm prosthesis. It was concluded that this new concept for prosthesis fitting of trans-humeral amputees looks promising, but alternative designs of the implant should be tested.

The rate of prosthetic joint infection is underestimated in the arthroplasty registers

In the present issue of Acta Orthopaedica, 2 studies have contributed results on different aspects of the rate of prosthetic joint infection (PJI). The studies have in common that they combined different patient registries, i.e. different data sources, to identify patients who had been reoperated due to PJI. In a study from Finland by Kaisa Huotari et al., the authors’ rationale for this approach was that incidence studies solely based on the Finnish Arthroplasty Register (FAR) had a tendency to underestimate the number of PJIs (Jamsen et al. 2009, Huotari et al. 2010). Thus, the authors combined data from FAR with data from the (Finnish) Hospital Discharge Register to study the rate of late PJI. The other study on PJI presented in this issue of Acta is from Denmark, by Per Hviid Gundtoft et al. The aim of that study was to estimate “the true incidence of surgically treated deep prosthetic joint infection”. By combining several data sources, including data on microbiology and blood tests, the authors have concluded that 40% of surgically treated PJIs were not reported to the Danish Hip Arthroplasty Register (DHR). Completeness of data when it comes to registration of primary hip and knee prostheses in the registers has been impressively high, in fact close to 100 percent. The failure endpoint in the registers has been any revision/reoperation of the prosthesis, and the completeness of revision data in general has also been considered to be high (Sodermann et al. 2000, Pedersen et al. 2004, Espehaug et al. 2006). It might therefore come as a surprise to the readers of this journal that only 60% of the surgically treated PJIs were reported to Danish Hip Arthroplasty Register. However, in its annual report in 2011 the Swedish Hip Arthroplasty Register (SHAR) presented the results of a study that showed that only 67% of surgically treated PJIs were reported to the SHAR (Lindgren et al. 2014). There could be different ways of explaining this under-registration of PJIs in the registers. In the report from the SHAR in 2011, it was stated that the degree to which the various hospitals reported their PJIs to the SHAR varied considerably. Many hospitals reported all their PJIs, whereas some hospitals reported less than 20%. As already mentioned, the failure endpoints in the arethroplasty registers have been revision/reoperation of the prosthesis. The register data are based on a notification form filled in by the operating surgeon immediately after the operation. As a means of registering all the PJIs that are treated operatively, the design of the notification form used in different countries has changed over time. In all the Nordic countries, minor prosthetic revision—which includes exchange of loose prosthetic parts, such as caput and liners—should be reported to the register, and also soft tissue debridement in cases of hip PJI. In Finland, the last revision of the notification form was done in 2014, while in Denmark minor revisions and soft tissue debridement have been options in the notification form for several years. This means that in the study period 2005–2011, all PJIs that were reoperated should have been registered in the Danish Arthroplasty Register (perhaps with the exception of reoperation due to a superficial infection). One could speculate on the degree to which the current practice of reporting to the register has been implemented by the different hospitals, and to what degree this has been followed up by the individual orthopedic surgeons. It should be remembered that during the first decade(s) after the arthroplasty registers were established, it took several years before it was well known in the orthopedic community that a procedure like a Girdlestone operation should be reported to the register. The Norwegian Arthroplasty Register (NAR) was established in 1987. 12–15 years later (1999–2002), one-third of hospitals (17/50) reported less than 50% of their “removal revisions” to the NAR (Espehaug et al. 2006). An important weakness of the arthroplasty registers is that they are not designed for registration of infections. In Finland, Denmark, and Norway, the surgeon fills in a form and—based on a subjective assessment—the surgeon decides whether or not the revision/reoperation is due to an infection. This is done immediately after the operation. In most cases (80%), the proof of an infection is culture of the causative microbe in tissue biopsies taken peroperatively. A conclusive microbiological result will usually not be available until 2–7 days after surgery, i.e. 2–7 days after the notification form has been sent to the register. Positive (or negative) culture results might come as a surprise, but once the revision diagnosis is reported to the to the register, it is (probably) never changed. It is even the policy of the Nordic Arthroplasty Register Association that a priority should be made not to change the primary revision diagnosis (personal communication). The Swedish Knee Arthroplasty Register submits medical records together with the notification form in cases of prosthetic revision, and the revision diagnosis is validated by personnel at the registry. To my knowledge, whether or not this policy has improved the completeness of registry data in general—and registration of PJIs in particular—has not been evaluated. In some cases, diagnosis of a PJI is difficult. There is an ongoing international collaboration to reach an agreement on objective criteria for the diagnosis of a PJI (Zmistowski et al. 2014). Viewing a PJI as something to be assessed by the orthopedic surgeon at the operating theater is an outdated diagnostic approach. In the study from Denmark, it is interesting to notice that visible joint purulence was not a very reliable criterion of infection. The diagnosis of a PJI should be based on objective criteria. Amongst these objective criteria, bacterial growth is most important. In some cases it is difficult to make a definite diagnosis, and the diagnosis should, if possible, be based on a multidisciplinary approach where specialists in infectious diseases and medical microbiology are included. For our readers, it is self-evident that infection is the most devastating complication to prosthetic surgery. If the trends continue with increasing rates of multiresistant bacteria and less effective antibiotics, infection would become a limiting factor for further development of prosthetic surgery. Each hospital should be part of a national surveillance program to ensure continuous survey of the rate of PJI. The diagnosis of PJI should be based on internationally accepted criteria, and if possible, diagnosis and treatment should be the result of a multidisciplinary approach. A spin-off product of such an approach and strategy would probably also be an improvement in the validity and completeness of the registration of PJI in the arthroplasty register. How the procedures for reporting of PJIs to the register should be revised to achieve such an improvement is beyond the scope of this editorial comment.