Aim. We used a polymerase chain reaction (PCR) lateral flow assay1) to rapidly diagnose joint infection. We evaluated the usefulness of multiplex-PCR (PCR lateral flow assay: PCR-LF) using detailed clinical data. Method. A total of 35 synovial fluid samples were collected from 26 patients in whom bacterial infection was suspected, including 22 from knee joints, 11 from hip joints, and 2 from other joints. After purifying the DNA from the samples, multiplex PCR targeting two MRSA-associated genes (femA and mecA) and the bacterial 16S rRNA gene was performed. Amplified gene fragments were specifically detected with DNA probes immobilized on stick devices through DNA-DNA hybridization and visualization, enabling diagnosis of MRSA, MSSA, MRCNS, gram-positive, and/or gram-negative bacterial infection. Genetic identification of bacteria by determining the 16S rRNA gene sequence was also performed using multiplex PCR-positive samples. Finally, the usefulness of our PCR-LF method was evaluated using detailed clinical data. Results. The results of PCR-LF were 9 gram-positive and 1 gram-negative bacterial infections. Eleven bacterial species were identified based on 16S rRNA gene sequences. Ten (90.9%)of the eleven samples (bacterial species) were identified using our PCR-LF. Five samples were detected in bacterial cultures; two are MSSA, one is Streptococcus agalactiae, one is Escherichia coli, one is Prevotella oralis. We diagnosed 6 samples as clinical infections. Therefore, the sensitivity and specificity of the culture tests were 83% and 100%, respectively, while for PCR-LF, these values were 83% and 83%. Conclusions. PCR-LF is highly sensitive and effective for the rapid diagnosis of joint infection; however, dead bacteria may also be detected. Moreover, because the target bacterial species are limited, clinical diagnosis based on the results of multiple examinations is necessary

Objective. Superior bone ingrowth and resistance to bacterial infection are ideal for orthopaedic implants. We compared new bone formation, strength of bone bonding, and infection rates between silicon nitride ceramic (Si3N4; abbreviated SiN), medical-grade PEEK (PEEK), and titanium (Ti) in rat calvariae. PEEK and Ti are used in spinal and arthroplasty implants respectively, while SiN is a non-oxide ceramic used in spinal implants for the past 4 years. Methods. Specimens of 10 mm × 10 mm by 1.75 mm size were implanted into experimental calvarial defects in 2-year old Wistar rats using standard surgical techniques (n's: SiN=48; PEEK=24; Ti=24). One group of animals was immediately inoculated with 1 × 104 Staphylococcus epidermidis; control animals received saline only. After sacrifice at 3 days, 7 days, 14 days, and 3 months post-inoculation (n=4 rats per time period), one calvarial sample each for PEEK and Ti, and two samples for SiN (per bacterial condition and time point) were retrieved for histology; remaining samples were used for sample push-out testing with a Micro Tester 5848 (Instron) with a 1-kN load cell, using published techniques. New bone formation was measured with tetracycline double-labeling at 11 and 4 days before the 14-day and 3-month time periods. Results. Of the materials tested, 3-month bone ingrowth and periprosthetic infection rates were most favorable for SiN (Fig. 1). At 90 days post-implantation without bacteria, new bone growth in calvariae with SiN was ∼69% compared with 24% and 36% for PEEK and Ti, respectively. With bacterial inoculation, new bone growth was 21%, 26% and 41% for PEEK, Ti, and SiN, respectively. At 3-months, live bacteria were observed for PEEK (88%) and Ti (21%), while no bacteria were present around SiN (Fig. 2). Push-out strength was greater for SiN when compared to Ti and PEEK (Fig. 3). Conclusions. Superior bone formation, bone ingrowth, and bacterial resistance were associated with SiN when compared to Ti and PEEK. SiN proved effective in inhibiting bacteria and promoting osteogenesis in experimental osseous defects, when compared to Ti and PEEK. These observations are most likely related to the hydrophilic properties of SiN that contributed to the adsorption of proteins known to decrease bacteria attachment and growth (vitronectin and fibronectin). We conclude that SiN may be a superior biomaterial for the development of orthopaedic implants

Aim. Galleria mellonella larvae is a well-known insect infection model that has been used to test the virulence of bacterial and fungal strains as well as for the high throughput screening of antimicrobial compounds against infections. Recently, we have developed insect infection model G. mellonella larvae to study implant associated biofilm infections using small K-wire as implant material. Here, we aimed to further expand the use of G. mellonella to test other materials such as bone cement with combination of gentamicin to treat implant-associated infections. Method. The poly methyl methacrylate (PMMA) with and without gentamicin and liquid methyl methacrylate (MMA) were kindly provided by Heraeus Medical GmbH, Wehrheim. To make the bone cement implants as cubes, Teflon plate (Karl Lettenbauer, Erlangen) with specified well size was used. The Radiopaque polymer and monomer were mixed well in a bowl, applied over on to the Teflon plate and pressed with spatula to form fine and uniform cubes. After polymerization, the bone cement implants were taken out of the Teflon well plate with the help of pin. For the infection process, bone cement cubes were pre-incubated with S. aureus EDCC 5055 culture at 5×10. 6. CFU/ml for 30 min at 150 rpm shaking conditions. Later, these implants were washed with 10ml PBS and implanted in the larvae as mentioned. Survival of the larvae were observed at 37°C in an incubator. To analyze the susceptibility of the bacterial infections towards gentamicin, survival of the larvae compared with control group implanted only with bone cement. The effect of gentamicin was also measured in terms of S. aureus load in larvae on 2. nd. day. SEM analysis was performed to see the effect of gentamicin on biofilm formation on bone cement. Results. Our experiments established the G. mellonella as an excellent model to screen bone cement with antimicrobial compounds against bacterial infections. The gentamicin bone cement samples showed excellent S. aureus bacterial load reduction after the implantation in G. mellonella model. The bone cement with gentamicin showed better survival of larvae infected with S. aureus compared to control. Finally, the gentamicin also affected the biofilm formation on the bone cement surface with S. aureus. Conclusions. Thus, our work showed G. mellonella is a rapid, cheap economical pre-clinical model to study the bone cement associate bacterial infections as well as screening of the various antimicrobial compounds

Aims. Bacterial infection activates neutrophils to release neutrophil extracellular traps (NETs) in bacterial biofilms of periprosthetic joint infections (PJIs). The aim of this study was to evaluate the increase in NET activation and release (NETosis) and haemostasis markers in the plasma of patients with PJI, to evaluate whether such plasma induces the activation of neutrophils, to ascertain whether increased NETosis is also mediated by reduced DNaseI activity, to explore novel therapeutic interventions for NETosis in PJI in vitro, and to evaluate the potential diagnostic use of these markers. Methods. We prospectively recruited 107 patients in the preoperative period of prosthetic surgery, 71 with a suspicion of PJI and 36 who underwent arthroplasty for non-septic indications as controls, and obtained citrated plasma. PJI was confirmed in 50 patients. We measured NET markers, inflammation markers, DNaseI activity, haemostatic markers, and the thrombin generation test (TGT). We analyzed the ability of plasma from confirmed PJI and controls to induce NETosis and to degrade in vitro-generated NETs, and explored the therapeutic restoration of the impairment to degrade NETs of PJI plasma with recombinant human DNaseI. Finally, we assessed the contribution of these markers to the diagnosis of PJI. Results. Patients with confirmed PJI had significantly increased levels of NET markers (cfDNA (p < 0.001), calprotectin (p < 0.001), and neutrophil elastase (p = 0.022)) and inflammation markers (IL-6; p < 0.001) in plasma. Moreover, the plasma of patients with PJI induced significantly more neutrophil activation than the plasma of the controls (p < 0.001) independently of tumour necrosis factor alpha. Patients with PJI also had a reduced DNaseI activity in plasma (p < 0.001), leading to a significantly impaired degradation of NETs (p < 0.001). This could be therapeutically restored with recombinant human DNaseI to the level in the controls. We developed a model to improve the diagnosis of PJI with cfDNA, calprotectin, and the start tail of TGT as predictors, though cfDNA alone achieved a good prediction and is simpler to measure. Conclusion. We confirmed that patients with PJI have an increased level of NETosis in plasma. Their plasma both induced NET release and had an impaired ability to degrade NETs mediated by a reduced DNaseI activity. This can be therapeutically restored in vitro with the approved Dornase alfa, Pulmozyme, which may allow novel methods of treatment. A combination of NETs and haemostatic biomarkers could improve the diagnosis of PJI, especially those patients in whom this diagnosis is uncertain. Cite this article: Bone Joint J 2024;106-B(9):1021–1030

Aim. While 16S rRNA PCR - Sanger sequencing has paved the way for the diagnosis of culture-negative bacterial infections, it does not provide the composition of polymicrobial infections. We aimed to evaluate the performance of the Nanopore-based 16S rRNA metagenomic approach using partial-length amplification of the gene, and to explore its feasibility and suitability as a routine diagnostic tool for bone and joint infections (BJI) in a clinical laboratory. Method. Sixty-two clinical samples from patients with BJI were sequenced on MinION* using the in-house partial amplification of the 16S rRNA gene. BJI were defined based on the ICM Philly 2018 and EBJIS 2021 criteria. Among the 62 samples, 16 (26%) were culture-positive, including 6 polymicrobial infections, and 46 (74%) were culture-negative from mono- and polymicrobial infections based on Sanger-sequencing. Contamination, background noise definition, bacterial identification, and time-effectiveness issues were addressed. Results. Results were obtained within one day. Setting a threshold at 1% of total reads overcame the background noise issue and eased interpretation of clinical samples. The partial 16S rRNA metagenomics approach had a greater sensitivity compared both to the culture method and the Sanger sequencing. All the 16 culture-positive samples were confirmed with the metagenomic sequencing. Bacterial DNA was detected in 32 culture-negative samples (70%), with pathogens consistent with BJI. The 14 Nanopore negative samples included 7 negative results confirmed after implementation of other molecular techniques and 7 false-negative MinION results: 3 Kingella kingae infections detected after targeted-PCR only, 2 Staphylococcus aureus infections and 2 Pseudomonas aeruginosa infections sterile on agar plate media and detected only after implementation of blood culture media, advocating for the very low inoculum. Conclusions. The results discriminated polymicrobial samples, and gave accurate bacterial identifications compared to Sanger-based results. They confirmed that Nanopore technology is user-friendly as well as cost- and time-effective. They also indicated that 16S rRNA targeted metagenomics is a suitable approach to be implemented for routine diagnosis of culture-negative samples in clinical laboratories. * Oxford Nanopore Technologies

Aim. Osteomyelitis is a difficult-to-treat disease with high chronification rates. The surgical amputation of the afflicted limb sometimes remains as the patients’ last resort. Several studies suggest an increase in mitochondrial fission as a possible contributor to the accumulation of intracellular reactive oxygen species and thereby to cell death of infectious bone cells. The aim of this study is to analyze the ultrastructural impact of bacterial infection and its accompanying microenvironmental tissue hypoxia on osteocytic and osteoblastic mitochondria. Method. 19 Human bone tissue samples from patients with osteomyelitis were visualized via light microscopy and transmission electron microscopy. Osteoblasts, osteocytes and their respective mitochondria were histomorphometrically analyzed. The results were compared to the control group of 5 non-infectious human bone tissue samples. Results. The results depicted swollen hydropic mitochondria including depleted cristae and a decrease in matrix density in the infectious samples as a common finding in both cell types. Furthermore, perinuclear clustering of mitochondria could also be observed regularly. Additionally, increases in relative mitochondrial area and number could be found as a sign for increased mitochondrial fission. Conclusions. The results show that mitochondrial morphology is altered during osteomyelitis in a comparable way to mitochondria from hypoxic tissues. This suggests that manipulation of mitochondrial dynamics in a way of inhibiting mitochondrial fission may improve bone cell survival and exploit bone cells regenerative potential to aid in the treatment of osteomyelitis

Aim. Treatment algorithms for fracture-related nonunion depend on the presence or absence of bacterial infection. However, the manifestation of septic nonunion varies. Low-grade infections, unlike manifest infections, lack clinical signs of infection and present similarly to aseptic nonunion. The clinical importance of low-grade infection in nonunion is not entirely clear. Therefore, the aim of this study was to evaluate the clinical relevance of low-grade infection in the development and management of femoral or tibial nonunion. Method. A prospective, multicenter clinical study enrolled patients with nonunion and regular healed fractures. Preoperatively, complete blood count without differential, C-reactive protein (CRP), and procalcitonin were obtained, clinical signs of infection were recorded, and a suspected septic or aseptic diagnosis was made based on history and clinical examination. During surgical nonunion revision or routine implant removal, tissue samples were collected for microbiology and histopathology, and osteosynthesis material for sonication. Nonunion patients were followed for 12 months. Definitive diagnosis of “septic” or “aseptic” nonunion was made according to diagnostic criteria for fracture-related infection, considering the results of any further revision surgery during follow-up. Results. 34 patients with regular healed fractures were included. 62 nonunion patients were diagnosed as aseptic, 22 with manifest, and 23 with low-grade infection. The positive predictive value was 88% and the negative predictive value 72% for the suspected diagnosis. The nonunion groups had significantly higher CRP levels than the regular healer group. Differentiation between septic and aseptic nonunion based on blood values was not possible. Low-grade infection demonstrated less frequently histopathologic signs of infection than manifest infection (22% vs. 50%, p=0.048), with 15% of regular healers having histopathologic signs of infection. Cutibacterium acnes was less present in manifest compared to low-grade infection (p=0.042). Healing rates for septic nonunion involving C. acnes were significantly lower for manifest infection (20%) than for low-grade infection (100%, p=0.002). Patients with low-grade infection were treated with systemic antibiotics less frequently than patients with manifest infection (p=0.026), with no significant difference in healing rate (83% vs. 64%), which was slightly lower for low-grade infection than for aseptic nonunion (90%). Conclusions. Low-grade infections play a significant role in nonunion development and are difficult to diagnose preoperatively due to the lack of clinical signs of infection and unremarkable blood counts. However, our results imply that for low-grade infections, antibiotic therapy may not always be mandatory to heal the nonunion. This study was supported by the German Social Accident Insurance (FF-FR0276)

Aim. A septic revision of an artificial joint is routinely split up in a so-called dirty phase and a clean phase. The measures taken to initiate the start of the clean phase vary significantly between musculoskeletal infection centers. We performed simulations of one-step exchanges of infected THAs and sought to 1) determine the effect of different clean phase protocols on the sterile field, and 2) determine whether or not it is possible to re-implant the new prosthesis completely clean. Method. Nine fresh frozen cadaveric hips were used and primary THA was undertaken via a direct anterior approach. Before implantation of the components varying amounts of fluorescent powder (GloGerm) were deposited, simulating bacterial infection. Second, a one-step exchange was performed via a posterolateral approach. After implant removal, debridement, and lavage, randomization determined which clean phase protocol was followed, i.e. no, some or full additional measures. Finally, the new prosthesis was re-implanted (fig. 1). In order to determine the effect of different clean phase protocols on contamination of the sterile field standardized UV light-enhanced photographs were obtained of 1) the gloves, 2) the instrument table, 3) the drapes, and 4) the wound and these were ranked on cleanliness by a blind panel of hip surgeons. In order to determine whether or not it is possible to re-implant the prosthesis completely clean, the implant was taken out again at the end of the one-step exchange and inspected for contamination under UV light. Results. The gloves, the instrument table, the drapes (fig. 2) and the wound were significantly cleaner after a clean phase using full additional measures compared to partial or no additional measures (p < 0.000). Partial measures were able to reduce some of the contamination of the gloves and the wound, but had no effect on the drapes and the instrument table. All re-implanted implants were contaminated with some amount of fluorescent powder at the end of the one-step exchange. Conclusions. We advise to incorporate a clean phase with full additional measures into the surgical treatment of prosthetic joint infections, as the effect of partial measures seems to be a poor compromise. The results of this study have now been published: Vles G, Bossen J, Kloos J, Debeer P, Ghijselings S. On the value and limitations of incorporating a “clean phase” into the surgical treatment of prosthetic joint infections - an illustrative cadaveric study using fluorescent powder. J Exp Orthop. 2022 Mar 21;9(1):28. doi: 10.1186/s40634-022-00467-x

Aim. Bone regeneration following the treatment of Staphylococcal bone infection or osteomyelitis is challenging due to the ability of Staphylococcus aureus to invade and persist within bone cells, which could possibly lead to antimicrobial tolerance and incessant bone destruction. Here, we investigated the influence of Staphylococcal bone infection on osteoblasts metabolism and function, with the underlying goal of determining whether Staphylococcus aureus-infected osteoblasts retain their ability to produce extracellular mineralized organic matrix after antibiotic treatment. Method. Using our in vitro infection model, human osteoblasts-like Saos-2 cells were infected with high-grade Staphylococcus aureus EDCC 5055 strain, and then treated with 8 µg/ml rifampicin and osteogenic stimulators up to 21-days. Results. Immunofluorescence and transmission electron microscopic (TEM) imaging demonstrated the presence of intracellular bacteria within the infected osteoblasts as early as 2 hours post-infection. TEM micrographs revealed intact intracellular bacteria with dividing septa indicative of active replication. The infected osteoblasts showed significant amounts of intracellular bacteria colonies and alteration in metabolic activity compared to the uninfected osteoblasts (p≤0.001). Treatment of S. aureus-infected osteoblasts with a single dose of 8 µg/ml rifampicin sufficiently restored the metabolic activity comparative to the uninfected groups. Alizarin red staining and quantification of the rifampicin-treated infected osteoblasts revealed significantly lower amount of mineralized extracellular matrix after 7-days osteogenesis (p<0.05). Interestingly, prolonged osteogenic stimulation and rifampicin-treatment up to 21 days improved the extracellular matrix mineralization level comparable to the rifampicin-treated uninfected group. However, the untreated (native) osteoblasts showed significantly more quantity of mineral deposits (p≤0.001). Ultrastructural analysis of the rifampicin-treated infected osteoblasts at 21-days osteogenesis revealed active osteoblasts and newly differentiated osteocytes, with densely distributed calcium crystal deposits within the extracellular organic matrix. Moreover, residual colony of dead bacteria bodies and empty vacuoles of the fully degraded bacteria embedded within the mineralized extracellular matrix. Gene expression level of prominent bone formation markers, namely RUNX2, COL1A1, ALPL, BMP-2, SPARC, BGLAP, OPG/RANKL showed no significant difference between the infected and uninfected osteoblast at 21-days of osteogenesis. Conclusions. Staphylococcus aureus bone infection can drastically impair osteoblasts metabolism and function. However, treatment with potent intracellular penetrating antibiotics, namely rifampicin restored the metabolic and bone formation activity of surviving osteoblasts. Delay in early osteogenesis caused by the bacterial infection was significantly improved over time after successful intracellular bacteria eradication

Aim. The coronavirus disease 2019 (COVID-19) pandemic presents significant challenges to healthcare systems globally. Orthopaedic surgeons are at risk of contracting COVID-19 due to their close contact with patients in both outpatient and theatre environments. The aim of this review was to perform a literature review, including articles of other coronaviruses, to formulate guidelines for orthopaedic healthcare staff. Methods. A search of Medline, EMBASE, the Cochrane Library, World Health Organization (WHO), and Centers for Disease Control and Prevention (CDC) databases was performed encompassing a variety of terms including ‘coronavirus’, ‘covid-19’, ‘orthopaedic’, ‘personal protective environment’ and ‘PPE’. Online database searches identified 354 articles. Articles were included if they studied any of the other coronaviruses or if the basic science could potentially applied to COVID-19 (i.e. use of an inactivated virus with a similar diameter to COVID-19). Two reviewers independently identified and screened articles based on the titles and abstracts. 274 were subsequently excluded, with 80 full-text articles retrieved and assessed for eligibility. Of these, 66 were excluded as they compared personal protection equipment to no personal protection equipment or referred to prevention measures in the context of bacterial infections. Results. There is a paucity of high quality evidence surrounding COVID-19. This review collates evidence from previous coronavirus outbreaks to put forward recommendations for orthopaedic surgeons during the COVID-19 pandemic. The key findings have been summarized and interpreted for application to the orthopaedic operative setting. Conclusion. For COVID-19 positive patients, minimum suggested PPE includes N95 respirator, goggles, face shield, gown, double gloves, and surgical balaclava. Space suits not advised. Be trained in the correct technique of donning and doffing PPE. Use negative pressure theatres if available. Minimize aerosolization and its effects (smoke evacuation and no pulse lavage). Minimize further unnecessary patient-staff contact (dissolvable sutures, clear dressings, split casts)

The role of biofilm in pathogenesis of several chronic human infections is widely accepted, as this structure leads pathogens to persist among the human body, being protected from the action of antibacterial molecules and drugs (1). It has been estimated that up to 65% of bacterial infections are caused by microorganisms growing in biofilms (2). Moreover, biofilm is involved in device-related orthopaedic bacterial infections, which are unaffected by vaccines and antibiotic therapies, constituting a serious problem for the human health care. The aim of the present work was to evaluate the anti-biofilm action of a selected and patented lactobacillus strain (MD1) supernatant, both on the in-formation- biofilm and on mature biofilm produced by pathogenic bacteria. MD1 was grown in BHI for 48 h at 37°C. After incubation, the sample was centrifuged for 5’ for 14,000 × g and the supernatant previously filtered and treated in order to obtain the anti-biofilm compounds (Special Supernatant – SS) was collected. Staphylococcus aureus and Pseudomonas aeruginosa strains were grown in BHI for 24h at 37°C. The anti-biofilm ability of the tested SS – lactobacillus strain was evaluated by a spectrophotometric method according to Christensen at al., following the incubation of pathogens and the “mature biofilm” with the lactobacillus supernatant. Confocal Laser Scanning Microscopy was used to confirm the data obtained from Crystal Violet Assay. After the incubation of the SS with pathogens and mature biofilm, the formation of biofilm was inhibited and a significant disruption of the mature biofilm was observed. Interestingly, the same properties were observed also when the SS pH was neutralized to pH 6.5. In particular, the reduction of biofilm production and the disruption of mature biofilm was about 50–70% for all microorganisms. The SS lactobacillus strain MD1 exhibited a relevant antibiofilm action against mature and in-formation-biofilm produced by S. aureus and P. aeruginosa strains tested in the study. Moreover, the antibiofilm action has been observed to be pH-independent, as when the supernatant was neutralized to pH 6.5, the reduction of pathogenic biofilm has been still observed. These promising results highlighted the possibility to use this SS-lactobacillus anti-biofilm property to develop a cost-effective and safety treatment able to reduce the impact of pathogenic biofilm on device-related orthopaedic bacterial infections

Aims. Periprosthetic fungal infections are rare and account for 1–2% of all periprosthetic joint infections (PJI). This study aims at presenting treatment details, clinical and microbiological results in a large single centre cohort. Methods. We retrospectively identified 29 patients (9 total knee replacements (TKA) and 20 total hip replacements (THA) treated for a fungal infection between 2007 and 2019. Microbiological findings, patient demographics and complications were analysed. Statistical analysis was performed using descriptive statistics; non-parametric analysis were performed using the Mann-Whitney U-Test. Infection-free survival was determined using Kaplan-Meier analysis and differences in survival were analysed using the log-rank test. The p value was set at p<0.05 with 95% confidence intervals (95% CI) provided. Results. 28% (8/29) suffered from reinfection. The reinfection-free survival probability was 65% (95% CI 45–85) after a median follow- up period of 28 months (IQR 6 – 39). With the numbers we had, we were not able to detect a difference between THA and TKA re-infections (p=0.517). Four patients underwent amputation, 3 patients had a definitive girdlestone hip and eight patients died after a median of 5 months after first-stage surgery (IQR 1–7). All patients treated had positive synovial fluid or tissue cultures for Candida species. In 22 /29 patients C. albicans, in 3 patients C. parapsilosis, in 2 patients C. glabrata and in 1 patient each C. famata, C. dubliniensis and C. gulliermondii. Polymicrobial bacterial infection was found in 86% of patients with staphylococci in 20 patients, E. coli in 2 patients, vancomycin-resistant enterococci, pseudomonas, acinetobacter and achromobacter species in 1 patient each. When investigating risk factors for reinfection, with the numbers we had we were not able to find a significant difference for patients with polymicrobial infection (p=0.974), azole-resistant Candida (p=0.491), tobacco users (p=0.175), or diabetics (p=0.54). Furthermore, median age (73 vs. 72, p=0.756) and Charlson comorbidity score (6 (interquartile range (IQR) 4–8) vs. 8 (IQR 5–10), p=0.184) were not different between the groups while on the other hand there was a trend for a higher body mass index in patients with reinfection (34 (IQR 31–38) vs. 28 (IQR 25–33), p=0.075). Conclusions. Fungal PJI is associated with poor reinfection free survival, frequent revisions, and high mortality. All infections were caused by Candida spp. in which azole-resistance most be considered when planning treatment. While polymicrobial infection complicated treatment there was no difference in survival. A higher BMI and comorbidity score might be associated with higher risk for reinfections

INTRODUCTION. Peri-prosthetic fungal infection is generally considered more difficult to cure than a bacterial infection. Two-stage exchange is considered the gold standard of surgical treatment. A recent study, however, reported a favorable outcome after one stage exchange in selected cases where the fungus was identified prior to surgery. The routine one stage exchange policy for bacterial peri-prosthetic infection involves the risk of identifying a fungal infection mimicking bacterial infection solely on intraoperative samples, i.e. after reimplantation, realizing actually a one stage exchange for fungal infection without pre-operative identification of the responsible fungus, which is considered to have a poor prognosis. We report two such cases of prosthetic hip and knee fungal infection. Despite this negative characteristic, no recurrence of the fungal infection was observed. CASE N°1: A 78 year old patient was referred for loosening of a chronically infected total hip arthroplasty (Staphylococcus aureus and Streptococcus dysgalactiae). One stage exchange was performed. Intraoperative bacterial cultures remained sterile. Two fungal cultures were positive for Candida albicans. Antifungal treatment was initiated for three months. No infection recurrence was observed at three year follow up. CASE N° 2: A 53-year-old patient was referred for loosening of a chronically infected total knee prosthesis (Staphylococcus aureus methicillin susceptible, Klebsiella pneumoniae and Staphylococcus epidermidis). One stage exchange was performed. Intraoperative bacterial cultures remained sterile. Five fungal cultures were positive for Candida albicans. Antifungal treatment was initiated for three months. No infection recurrence was observed at two-year follow-up. DISCUSSION. This experience suggests that eradication of fungal infection of a total hip or knee arthroplasty may be possible after one stage exchange even in cases where the diagnosis of fungal infection was not known before surgery, when the fungus was not identified and its antifungal susceptibility has not been evaluated before surgery. It is however not possible to propose this strategy as a routine procedure. CONCLUSION. We suggest evaluating the results of one stage exchange for peri-prosthetic fungal infection on a larger scale

Aim. Septic arthritis (SA) is considered a medical emergency. The most common etiological agents are glucose consuming bacteria, so we evaluated the clinical utility of synovial fluid (SF) glucose levels and other biochemical parameters for supporting the diagnosis of the disease and their association with a positive bacteria culture and joint destruction. Methods. Adult patients with SA diagnose were enrolled prospectively between July 2018 and October 2019. As control group, adults with knee osteoarthritis, meniscus and/or knee ligaments lesions were enrolled. SF samples were obtained from the joints by arthrocentesis/arthrotomy. Microbiological analyses of SF were performed using Brucella broth blood culture flasks, samples were incubated at 37°C with 5% CO. 2. for 24 hours. Gram stain, chocolate and blood agar were used for the identification and growth of the bacteria. SF glucose levels, pH and leukocyte esterase were measured as biochemical parameters using a glucometer and colorimetric test strips. The Outerbridge classification was used for grading the osteochondral injury. Furthermore, blood samples were collected from patients and control subjects for determining glucose levels. Results. We included 8 subjects with knee ligaments lesions, 6 with meniscus lesions and 5 with osteoarthritis as control group, as well as 20 patients with SA diagnose. The mean age of the patients was 57.8 years with a 65% of male predominance. The most common affected joint was the knee (85%). SF culture was positive in 60% of the cases and the most common etiological agent was Staphylococcus aureus (58.3%). SF glucose levels from patients were lower than the controls (P=0.0018) and showed the lowest concentration in patients with a positive culture (P=0.0004). There was also a difference between blood and SF glucose concentration from the positive culture patients (P<0.0001). Leucocyte esterase presented the highest values in positive culture patients (P=<0.0001) and a more acidic pH was found compared to the control group (P<0.0001). Regarding the osteochondral injury, the lowest concentrations of SF glucose were found in patients with a higher grade in the classification (P = 0.0046). Conclusions. SF glucose and leukocyte esterase concentrations might be a quick and cheap useful parameter for the physician for distinguishing between bacterial infection and not infected joint. In addition, the lowest SF glucose levels might give information about the joint damage due to the disease

Background:. Early diagnosis of septic arthritis and osteomyelitis in children is essential to prevent long term sequelae. The diagnosis for these orthopaedic emergencies can be difficult and challenging especially in infants. Standard blood tests used for diagnosis have a low specificity. Procalcitonin (PCT) is significantly elevated in bacterial infections and remains low in viral infections and inflammatory conditions. Good positive predictive values for PCT have been obtained in various studies used in paediatric infections, but limited studies have examined the role in orthopaedic infections. Aims:. To introduce PCT testing in the work up of Septic Arthritis (SA) and Acute Osteomyelitis (OM) and to see if the test is useful in the diagnosis. Also to determine whether 0.2 ng/ml is a suitable cutoff level as indicated by previous studies. Method:. All children under 14 years presenting with signs and symptoms of SA/OM from 1 June 2009 to 31 June 2010 were subjected to standard blood tests with addition of PCT and compared to a control group. The definitive diagnosis was made by microbiologic examination obtained in theatre. A PCT cut-off level of 0.2 ng/mL was used. Results:. A total number of 33 patients were included in the study. Eight patients were diagnosed as OM, 4 as SA and 21 had another diagnosis. Staphylococcus aureus was the most common organism isolated in this series with no resistant organisms seen. In the SA/OM Group 11 of the 12 patients had an increased PCT level and 4 in the other diagnosis group had raised PCT. The calculated sensitivity of PCT was 92% with a confidence interval of 62–100% and the specificity was 81% with a confidence interval of 58–95%. In this study the sensitivity of CRP was 100% while the specificity 26%. The positive predictive value for PCT in this study was 73% and the negative predictive value was 94%. The accuracy for PCT in Septic Arthritis and Osteomyelitis in this study was 85%. Conclusions:. The calculated sensitivity and specificity in this study has shown that PCT testing can aid in the diagnosis of SA/OM in children using 0.2 ng/ml as cut-off level. PCT was more specific for bacterial infections in this study than CRP. Further research is needed with larger numbers to conclusively prove that this specific cut-off for PCT is significant

Aim. Virulent bacteriophages are known to be an effective therapy against various human bacterial infections. The aims of the study are to evaluate i) the killing activity of an antistaphylococcal phage lysate (ASPL), available in the Czech Republic for topical application, against Staphylococcal aureus (Sa) strains isolated in orthopedic infections; ii) the antimicrobial activity of ASPL against biofilm-embedded cells of a methicillin-resistant Sa (MRSA) standard strain. Method. The susceptibility of 25 MRSA and 18 methicillin-sensitive Sa (MSSA) strains to the ASPL was evaluated by spot assay. In addition, susceptibility of four laboratory MRSA strains, including ATCC 43300, ATCC 33591, Mu3 (MRSA/hetero vancomycin intermediate resistant Sa) and Mu50 (MRSA/vancomycin-resistant Sa) was also tested. The activity of ASPL against planktonic and biofilm-embedded MRSA ATCC 43300 was evaluated in real-time by isothermal microcalorimetry. The minimum heat inhibitory concentrations (MHIC) was defined as the lowest antimicrobial concentration leading to the lack of heat flow production after 24h for both planktonic and biofilm-embedded cells. The viability of bacterial cells was assessed by plating and colony counting. The minimum bactericidal concentration (MBC) was defined as the lowest antimicrobial concentration leading the reduction of 3 log CFU compared to the untreated control. Results. Around 34 out of 43 (79%) Sa strains were susceptible to the ASPL, including 17 MRSA and 17 MSSA strains. Both Mu3 and Mu50 (vancomycin intermediate and resistant MRSA, respectively) strains were also susceptible. Microcalorimetric evaluation of the activity of ASPL against planktonic cells of MRSA ATCC 43300 revealed the MHIC and the MBC were 10. 4. PFU/ml and 10. 5. PFU/ml, respectively. ASPL tested at 10. 5. PFU/ml was able to suppress the heat produced by biofilm bacterial cells, although this titer was not able to completely eradicate MRSA biofilm. Conclusions. ASPL showed a broad host spectrum among MRSA and MSSA strains associated with infections on implants, including strains that are resistant to vancomycin as well. ASPL exhibits a lytic activity against planktonic and biofilm MRSA and a titer of phage higher than 10. 5. PFU/ml is needed in order to achieve a complete eradication of MRSA biofilm. In conclusion, the antistaphylococcal phage lysate shows an excellent potential treatment of implant-related infections caused by Sa strains

Aim. Surgical site infections caused by Staphylococcus aureus (S. aureus) are associated with considerable clinical and economic burden. Studies assessing this burden in Germany have been limited to specific institutions, selected patient groups or not specific to S. aureus infections (SAI). This study was undertaken to further understand the burden of SAI following orthopedic surgeries in Germany. Method. All patients with at least one spine, endoprosthetic hip or knee surgery between 2012 and 2015 captured in the AOK PLUS claims database were included in this analysis. SAI were identified using S. aureus-specific ICD-10 codes following surgery. Exclusion criteria included: younger than 18, SAI in the 90 days preceding index, any surgery in the 180 days preceding index, surgery at the same body location as index in the preceding 365 days, or more than one surgery of interest during index hospitalization. Cumulative incidence and incidence density were used to assess SAI. Mortality, healthcare resource utilization and costs were compared between SAI and non-SAI group during the 1year follow-up post index surgery. Multivariate analyses were conducted while controlling for sex, age, Charlson Comorbidity Index (CCI), location of surgery, length of index hospitalization, recent fractures, other bacterial infections during index hospitalization and outpatient prescriptions for antibiotics in the year pre-index. Results. In total, 74,327 patients were included who underwent a knee (21,285), hip (29,429), or spine surgery (23,613). Mean age was 69.6 years, 61.6% were female and the mean CCI was 2.3. The SAI incidence post-orthopedic surgery was 20.2 cases per 1,000 patient-years within 1 year of index hospitalization; the cumulative incidence was 1.9%. Knee surgeries were associated with lower SAI risk compared to hip surgeries (HR=0.8; p=0.024), whereas spine surgeries did not differ significantly. Compared to non-SAI group, the SAI group had on average 4.4 times the number of hospitalizations (3.1 vs. 0.7) and 7.7 times the number of hospital days (53.5 vs. 6.9), excluding the index hospitalization (p-values<0.001). One year post-orthopedic mortality was 22.38% in the SAI and 5.31% in the non-SAI group (p<0.001). The total medical costs were significantly different between SAI and non-SAI groups (42,834€ vs. 13,781€; p<0.001). Adjusting for confounders, the SAI group had nearly 2 times the all-cause direct healthcare costs (exp(b)=1.9; p<0.001); and 2.5 times the risk of death (OR=2.5; p<0.001) compared to the non-SAI group. Conclusions. S. aureus infection risk after orthopedic surgeries persists and is associated with significant economic burden and risk of mortality

Background. One of the serious postoperative complications associated with joint replacement is bacterial infection. In our recent investigations, iodine supported titanium implants demonstrated antibacterial activity in both in vitro studies and clinical trials. But it is not clear whether iodine treated titanium implants produce strong bonding to bone. This study evaluated the bone bonding ability of titanium implants with and without iodine surface treatments. Methods. Titanium rods were implanted in intramedullary rabbit femur models, in regard to the cementless hip stem. The implant rods were 5mm in diameter and 25mm in length. Half of the implants were treated with iodine (ID implants) and the other half were untreated (CL implants). The rods were inserted into the distal femur; ID implants into the right femur and CL implants into the left. We assessed the bonding strength by a measuring pull-out test at 4, 8, and 12 weeks after implantation. The bone-implant interfaces were evaluated at 4 weeks after implantation. Results. Pull-out test results of the ID implants were 202, 355, and 344 N, at 4, 8, and 12 weeks, respectively, significantly higher than those of the CL implants (102, 216, and 227 N). Histological examination revealed that new bone formed on the surface of both types of implants, but significantly more bone made direct contact with the surfaces of the ID implants. Conclusion. This research showed that new type of coating, iodine coated titanium has low toxicity and good osteoconductivity

Few studies have been reported focusing on developing implant surface nanofiber (NF) coating to prevent infection and enhance osseointegration by local drug release. In this study, coaxial doxycycline (Doxy)-doped polycaprolactone/polyvinyl alcohol (PCL/PVA) NFs were directly deposited on the titanium (Ti) implant surface during electrospinning. The bonding strength of Doxy-doped NF coating on Ti implants was confirmed by a stand single-pass scratch test. The improved implant osseointegration by PCL/PVA NF coatings in vivo was confirmed by scanning electron microscopy, histomorphometry and micro computed tomography at 2, 4 and 8 weeks after implantation. The bone contact surface (%) changes of NF coating group (80%) is significantly higher than that of no NF group (< 5%, p<0.05). Finally, we demonstrated that Doxy-doped NF coating effectively inhibited bacterial infection and enhanced osseointegration in an infected (Staphylococcus aureus) tibia implantation rat model. Doxy released from NF coating inhibited bacterial growth up to 8 weeks in vivo. The maximal push-in force of Doxy-NF coating (38 N) is much higher than that of NF coating group (6.5 N) 8 weeks after implantation (p<0.05), which was further confirmed by quantitative histological analysis and micro computed tomography. These findings indicate that coaxial PCL/PVA NF coating doped with Doxy and/or other drugs have great potential in enhancing implant osseointegration and preventing infection

Aim. Despite the expanding research focusing on bacterial biofilm formation, specific histochemical biofilm stains have not been developed for light microscopy. Therefore, pathologists are often not aware of the presence of biofilm formation when examining slides for diagnosing bacterial infections, including orthopaedic infections. The aim of the present study was to develop a combined histochemical and immunohistochemical biofilm stain for simultaneous visualization of Staphylococcus aureus bacteria and extracellular matrix in different colours using light microscopy. Methods. Infected bone tissue was collected from two different porcine models of osteomyelitis inoculated with the biofilm forming S. aureus strain S54F9. The infection time was 5 and 15 days, respectively. First, 25 common histochemical protocols were used in order to find stains that could identify extracellular biofilm matrix. Hereafter, the histochemical protocols for Alcian Blue pH3, Luna and Methyl-pyronin green were combined with an immunohistochemical protocol based on a specific antibody against S. aureus. Finally, the three new combined protocols were applied to infected bone tissue from a child suffering from chronic staphylococcal osteomyelitis for more than a year. For all combined protocols applied on all types of tissue (porcine and human) the number of double stained bacterial aggregates were counted. On the same sections the percentage of extracellular matrix of representative bacterial aggregates was calculated by image analysis. Results. Simultaneous visualization of bacterial cells and extracellular matrix in different colours was detected in both porcine and human tissue sections with all three combined protocols. The bacterial cells were red to light brown and the extracellular matrix either light blue, blue or orange depending on the histochemical stain i.e. if it was Alcian blue pH3 (colouring polysaccharides), Luna or Methyl green-pyronin (both colouring extracellular DNA), respectively. In the porcine models, 10 percent of the bacterial aggregates in a 10× magnification field revealed both the extracellular matrix and bacteria simultaneously in two different colours. For the human case, this was seen in 90 percent of the bacterial aggregates. The percentage of extracellular matrix of representative bacterial aggregates was 60 and 20 percent in the human and porcine tissues, respectively. Conclusions. The amount of S. aureus biofilm extracellular matrix increased with infection time. A combination of histochemical and immunohistochemical staining is a practical method for identification and evaluation of S. aureus biofilm in orthopaedic infections