Aims. Fungal and mycobacterial periprosthetic joint infections (PJI) are rare events. Clinicians are wary of missing these diagnoses, often leading to the routine ordering of fungal and mycobacterial cultures on periprosthetic specimens. Our goal was to examine the utility of these cultures and explore a modern bacterial culture technique using bacterial blood culture bottles (BCBs) as an alternative. Methods. We performed a retrospective review of patients diagnosed with hip or knee PJI between 1 January 2010 and 31 December 2019, at the Mayo Clinic in Rochester, Minnesota, USA. We included patients aged 18 years or older who had fungal, mycobacterial, or both cultures performed together with bacterial cultures. Cases with positive fungal or mycobacterial cultures were reviewed using the electronic medical record to classify the microbiological findings as representing true infection or not. Results. There were 2,067 episodes of PJI diagnosed within the study period. A total of 3,629 fungal cultures and 2,923 mycobacterial cultures were performed, with at least one of these performed in 56% of episodes (n = 1,157). Test positivity rates of fungal and mycobacterial cultures were 5% (n = 179) and 1.2% (n = 34), respectively. After a comprehensive review, there were 40 true fungal and eight true mycobacterial PJIs. BCB were 90% sensitive in diagnosing true fungal PJI and 100% sensitive in detecting rapidly growing mycobacteria (RGM). Fungal stains were performed in 27 true fungal PJI but were only positive in four episodes (14.8% sensitivity). None of the mycobacterial stains was positive. Conclusion. Routine fungal and mycobacterial stains and cultures should not be performed as they have little clinical utility in the diagnosis of PJI and are associated with significant costs. Candida species and RGM are readily recovered using BCB. More research is needed to predict rare non-Candida fungal and slowly growing mycobacterial PJI that warrant specialized cultures. Cite this article: Bone Joint J 2022;104-B(1):53–58

Aims. Metagenomic next-generation sequencing (mNGS) is useful in the diagnosis of infectious disease. However, while it is highly sensitive at identifying bacteria, it does not provide information on the sensitivity of the organisms to antibiotics. The purpose of this study was to determine whether the results of mNGS can be used to guide optimization of culture methods to improve the sensitivity of culture from intraoperative samples. Methods. Between July 2014 and October 2019, patients with suspected joint infection (JI) from whom synovial fluid (SF) was obtained preoperatively were enrolled. Preoperative aspirated SF was analyzed by conventional microbial culture and mNGS. In addition to samples taken for conventional microbial culture, some samples were taken for intraoperative culture to optimize the culture method according to the preoperative mNGS results. The demographic characteristics, medical history, laboratory examination, mNGS, and culture results of the patients were recorded, and the possibility of the optimized culture methods improving diagnostic efficiency was evaluated. Results. A total of 56 cases were included in this study. There were 35 cases of JI and 21 cases of non-joint infection (NJI). The sensitivity, specificity, and accuracy of intraoperative microbial culture after optimization of the culture method were 94.29%, 76.19%, and 87.5%, respectively, while those of the conventional microbial culture method were 60%, 80.95%, and 67.86%, respectively. Conclusion. Preoperative aspirated SF detected via mNGS can provide more aetiological information than preoperative culture, which can guide the optimization and improve the sensitivity of intraoperative culture. Cite this article: Bone Joint J 2021;103-B(1):39–45

Aims. Use of molecular sequencing methods in periprosthetic joint infection (PJI) diagnosis and organism identification have gained popularity. Next-generation sequencing (NGS) is a potentially powerful tool that is now commercially available. The purpose of this study was to compare the diagnostic accuracy of NGS, polymerase chain reaction (PCR), conventional culture, the Musculoskeletal Infection Society (MSIS) criteria, and the recently proposed criteria by Parvizi et al in the diagnosis of PJI. Methods. In this retrospective study, aspirates or tissue samples were collected in 30 revision and 86 primary arthroplasties for routine diagnostic investigation for PJI and sent to the laboratory for NGS and PCR. Concordance along with statistical differences between diagnostic studies were calculated. Results. Using the MSIS criteria to diagnose PJI as the reference standard, the sensitivity and specificity of NGS were 60.9% and 89.9%, respectively, while culture resulted in sensitivity of 76.9% and specificity of 95.3%. PCR had a low sensitivity of 18.4%. There was no significant difference based on sample collection method (tissue swab or synovial fluid) (p = 0.760). There were 11 samples that were culture-positive and NGS-negative, of which eight met MSIS criteria for diagnosing infection. Conclusion. In our series, NGS did not provide superior sensitivity or specificity results compared to culture. PCR has little utility as a standalone test for PJI diagnosis with a sensitivity of only 18.4%. Currently, several laboratory tests for PJI diagnosis should be obtained along with the overall clinical picture to help guide decision-making for PJI treatment. Cite this article: Bone Joint J 2021;103-B(1):26–31

Aims. During revision procedures for aseptic reasons, there remains a suspicion that failure may have been the result of an undetected subclinical infection. However, there is little evidence available in the literature about unexpected positive results in presumed aseptic revision spine surgery. The aims of our study were to estimate the prevalence of unexpected positive culture using sonication and to evaluate clinical characteristics of these patients. Patients and Methods. All patients who underwent a revision surgery after instrumented spinal surgery at our institution between July 2014 and August 2016 with spinal implants submitted for sonication were retrospectively analyzed. Only revisions presumed as aseptic are included in the study. During the study period, 204 spinal revisions were performed for diagnoses other than infection. In 38 cases, sonication cultures were not obtained, leaving a study cohort of 166 cases. The mean age of the cohort was 61.5 years (. sd. 20.4) and there were 104 female patients. Results. Sonication cultures were positive in 75 cases (45.2%). Hardware failure was the most common indication for revision surgery and revealed a positive sonication culture in 26/75 cases (35%) followed by adjacent segment disease (ASD) in 23/75 cases (30%). Cutibacterium acnes and Staphylococcus epidermidis were the most commonly isolated microorganisms, observed in 45% and 31% of cases, respectively. C. acnes was isolated in 65.2% of cases when the indication for revision surgery was ASD. Conclusion. Infection must always be considered as a possibility in the setting of spinal revision surgery, especially in the case of hardware failure, regardless of the lack of clinical signs. Sonication should be routinely used to isolate microorganisms adherent to implants. Cite this article: Bone Joint J 2019;101-B:621–624

Aims. The aim of this study was to identify the incidence of positive cultures during the second stage of a two-stage revision arthroplasty and to analyse the association between positive cultures and an infection-free outcome. Patients and Methods. This single-centre retrospective review of prospectively collected data included patients with a periprosthetic joint infection (PJI) of either the hip or the knee between 2013 and 2015, who were treated using a standardised diagnostic and therapeutic algorithm with two-stage exchange. Failure of treatment was assessed according to a definition determined by a Delphi-based consensus. Logistic regression analysis was performed to assess the predictors of positive culture and risk factors for failure. The mean follow-up was 33 months (24 to 48). Results. A total of 163 two-stage revision arthroplasties involving 84 total hip arthroplasties (THAs) and 79 total knee arthroplasties (TKAs) were reviewed. In 27 patients (16.6%), ≥ 1 positive culture was identified at re-implantation and eight (29.6%) of these subsequently failed compared with 20 (14.7%) patients who were culture-negative. The same initially infecting organism was isolated at re-implantation in nine of 27 patients (33.3%). The organism causing re-infection in none of the patients was the same as that isolated at re-implantation. The risk of the failure of treatment was significantly higher in patients with a positive culture (odds ratio (OR) 1.7; 95% confidence interval (CI) 1.0 to 3.0; p = 0.049) and in patients with a higher Charlson Comorbidity Index (OR 1.5; 95% CI 1.6 to 1.8; p = 0.001). Conclusion. Positive culture at re-implantation was independently associated with subsequent failure. Surgeons need to be aware of this association and should consider the medical optimisation of patients with severe comorbidities both before and during treatment. Cite this article: Bone Joint J 2017;99-B:1490–5

Allografts of bone from the femoral head are often used in orthopaedic procedures. Although the donated heads are thoroughly tested microscopically before release by the bone bank, some surgeons take additional cultures in the operating theatre before implantation. There is no consensus about the need to take these cultures. We retrospectively assessed the clinical significance of the implantation of positive-cultured bone allografts. The contamination rate at retrieval of the allografts was 6.4% in our bone bank. Intra-operative cultures were taken from 426 femoral head allografts before implantation; 48 (11.3%) had a positive culture. The most frequently encountered micro-organism was coagulase-negative staphylococcus. Deep infection occurred in two of the 48 patients (4.2%). In only one was it likely that the same micro-organism caused the contamination and the subsequent infection. In our study, the rate of infection in patients receiving positive-cultured allografts at implantation was not higher than the overall rate of infection in allograft surgery suggesting that the positive cultures at implantation probably represent contamination and that the taking of additional cultures is not useful

We compared the use of broth culture medium for samples taken in theatre with the standard practice of placing tissue samples in universal containers. A total of 67 consecutive patients had standard multiple samples of deep tissue harvested at surgery and distributed equally in theatre either to standard universal containers or to broth culture medium. These samples were cultured by direct and enrichment methods. The addition of broth in theatre to standard practice led to an increase in sensitivity from 83% to 95% and an increase in negative predictive value from 77% to 91%. Placing tissue samples directly into broth in the operating theatre is a simple, inexpensive way to increase the sensitivity of cultures from infected patients, and does not appear to compromise the specificity of these cultures. Cite this article: Bone Joint J 2014;96-B:1566–70

We determined the rate of contamination of donated femoral heads at primary hip arthroplasty within a single region between July 1992 and July 2001. We established the null hypothesis that culture results played no role in predicting early failure of the joint because of infection. The rate of contamination was 9%. A positive culture, at the time of retrieval, was found in 367 of 4045 femoral heads. Coagulase-negative staphylococcus was isolated in 77% of the positive cases. At a minimum follow-up of one year, there was no statistically significant difference in the rate of complications or of revision of age-matched patients whose femoral heads had a positive culture compared with those whose femoral heads were sterile. Our findings confirm that culture of the femoral head plays no part in determining future failure of joint replacement in the donor

Bovine and human articular chondrocytes were seeded in 2% alginate constructs and cultured for up to 19 days in a rotating-wall-vessel (RWV) and under static conditions. Culture within the RWV enhanced DNA levels for bovine chondrocyte-seeded constructs when compared with static conditions but did not produce enhancement for human cells. There was a significant enhancement of glycosaminoglycans and hydroxyproline synthesis for both bovine and human chondrocytes. In all cases, histological analysis revealed enhanced Safranin-O staining in the peripheral regions of the constructs compared with the central region. There was an overall increase in staining intensity after culture within the RWV compared with static conditions. Type-II collagen was produced by both bovine and human chondrocytes in the peripheral and central regions of the constructs and the staining intensity was enhanced by culture within the RWV. A capsule of flattened cells containing type-I collagen developed around the constructs maintained under static conditions when seeded with either bovine or human chondrocytes, but not when cultured within the RWV bioreactor

Fresh-frozen allograft bone is frequently used in orthopaedic surgery. We investigated the incidence of allograft-related infection and analysed the outcomes of recipients of bacterial culture-positive allografts from our single-institute bone bank during bone transplantation. The fresh-frozen allografts were harvested in a strict sterile environment during total joint arthroplasty surgery and immediately stored in a freezer at -78º to -68º C after packing. Between January 2007 and December 2012, 2024 patients received 2083 allografts with a minimum of 12 months of follow-up. The overall allograft-associated infection rate was 1.2% (24/2024). Swab cultures of 2083 allografts taken before implantation revealed 21 (1.0%) positive findings. The 21 recipients were given various antibiotics at the individual orthopaedic surgeon’s discretion. At the latest follow-up, none of these 21 recipients displayed clinical signs of infection following treatment. Based on these findings, we conclude that an incidental positive culture finding for allografts does not correlate with subsequent surgical site infection. Additional prolonged post-operative antibiotic therapy may not be necessary for recipients of fresh-frozen bone allograft with positive culture findings. Cite this article: Bone Joint J 2015;97-B:427–31

We compared the changes in the ratio of type-I and type-II collagen in monolayer cultures of human articular chondrocytes (HAC). HAC were isolated from samples of cartilage from normal joints and cultivated in monolayer for up to 46 days. Expression of collagen type-I and type-II was determined by immunocytochemistry, Western blotting, and the nested reverse transcription polymerase chain reaction (RT-PCR), and quantified by real-time PCR. The transition from a spherical morphology to the flattened morphology of an anchorage-dependent culture was accompanied by a rapid change in the collagen phenotype with the replacement of collagen type II by collagen type I. This was confirmed by immunocytochemistry and Western blotting between days 21 and 28. Using techniques for the analysis of gene transcription (nested RT-PCR and real-time PCR), a complete switch of collagen gene expression was not observed. Expression of collagen type I increased 100-fold during the culture time. That of collagen type II was found during the entire period and decreased more than 100-fold. The main finding was that expression of the genes encoding collagen type I and II was highly time-dependent and the ratio of collagen type II to I (CII/CI), defined as an index of cell differentiation, was significantly higher (215- to 480-fold) at the beginning of the culture. At the end of the experimental culture time, ratios between 0.1 and 1 were reached

Colchicine is often used in the treatment of diseases such as familial Mediterranean fever (FMF) and gout. We have previously reported that patients with FMF who had colchicine on a daily basis and who had a total hip arthroplasty showed no heterotopic ossification after surgery. The mechanism by which colchicine causes this clinical phenomenon has never been elucidated. We therefore evaluated the effect of various concentrations of colchicine on cell proliferation and mineralisation in tissue culture, using rat and human cells with and without osteogenic potential. Cell proliferation was assessed by direct cell counts and uptake of (. 3. H)thymidine, and mineralisation by measuring the amount of staining by Alizarin Red. Our findings indicate that concentrations of colchicine of up to 3 ng/ml did not affect cell proliferation but inhibition was observed at 10 to 30 ng/ml. Mineralisation decreased to almost 50%, which was the maximum inhibition observed, at concentrations of colchicine of 2.5 ng/ml. These results indicate that colchicine at low concentrations, of up to 3 ng/ml, has the capacity to inhibit selectively bone-like cell mineralisation in culture, without affecting cell proliferation. Further clinical and laboratory studies are necessary to evaluate the effects of colchicine on biological processes involving the proliferation of osteoblasts and tissue mineralisation in vivo, such as the healing of fractures, the formation of heterotopic bone and neoplastic bone growth

In 78 consecutive patients, we performed fine-needle aspiration of the hip before revision surgery. At the revision operation biopsies were taken from the capsule and joint tissues. The aspirate and the homogenised soft-tissue specimens were cultured using a radiometric technique. Fifteen hips proved to be infected and 63 sterile on culture of the tissues taken during revision surgery; 94% of the aspiration results were correct. The sensitivity of the technique was 87% and the specificity 95%. Fine-needle aspiration combined with a radiometric culture technique is a simple and reliable method of diagnosing infection of hip prostheses

Wear debris was extracted from 21 worn hip and knee replacements. Its mutagenic effects were tested on human cells in tissue culture using the micronucleus assay and fluorescent in situ hybridisation. The extracted wear debris increased the level of micronuclei in a linear dose-dependent manner but with a tenfold difference between samples. The concentration of titanium +/− vanadium and aluminium within the wear debris was linearly related both to the level of centromere-positive micronuclei in tissue culture, indicating an aneuploid event, and to the level of aneuploidy in vivo in peripheral blood lymphocytes. The concentration of cobalt and chromium +/− nickel and molybdenum in the wear debris correlated with the total index of micronuclei in tissue culture, both centromere-positive and centromere-negative i.e. both chromosomal breakage and aneuploidy events. The results show that wear debris can damage chromosomes in a dose-dependent manner which is specific to the type of metal. The results from studies in vitro correlate with those in vivo and suggest that the wear debris from a worn implant is at least partly responsible for the chromosomal damage which is seen in vivo

Peri-tendinous injection of local anaesthetic, both alone and in combination with corticosteroids, is commonly performed in the treatment of tendinopathies. Previous studies have shown that local anaesthetics and corticosteroids are chondrotoxic, but their effect on tenocytes remains unknown. We compared the effects of lidocaine and ropivacaine, alone or combined with dexamethasone, on the viability of cultured bovine tenocytes. Tenocytes were exposed to ten different conditions: 1) normal saline; 2) 1% lidocaine; 3) 2% lidocaine; 4) 0.2% ropivacaine; 5) 0.5% ropivacaine; 6) dexamethasone (dex); 7) 1% lidocaine+dex; 8) 2% lidocaine+dex; 9) 0.2% ropivacaine+dex; and 10) 0.5% ropivacaine+dex, for 30 minutes. After a 24-hour recovery period, the viability of the tenocytes was quantified using the CellTiter-Glo viability assay and fluorescence-activated cell sorting (FACS) for live/dead cell counts. A 30-minute exposure to lidocaine alone was significantly toxic to the tenocytes in a dose-dependent manner, but a 30-minute exposure to ropivacaine or dexamethasone alone was not significantly toxic.

Dexamethasone potentiated ropivacaine tenocyte toxicity at higher doses of ropivacaine, but did not potentiate lidocaine tenocyte toxicity. As seen in other cell types, lidocaine has a dose-dependent toxicity to tenocytes but ropivacaine is not significantly toxic. Although dexamethasone alone is not toxic, its combination with 0.5% ropivacaine significantly increased its toxicity to tenocytes. These findings might be relevant to clinical practice and warrant further investigation.

We cultured human osteoblasts from trabecular bone explants and confirmed their phenotype by alkaline phosphatase assay, increased cyclic adenosine monophosphate production in response to prostaglandin E2 and radiographic micro-analysis of nodules of calcification. The osteoblasts were seeded on to demineralised human bone fragments and examined at ten-day intervals over a 50-day period by scanning electron microscopy. During this time the bank bone became progressively repopulated by the cultured osteoblasts. This system may offer a means of graft enhancement in elective orthopaedic and maxillofacial surgery by delivery of cultured autologous human osteoblasts to bone defects.

Aims. Accurate diagnosis of chronic periprosthetic joint infection (PJI) presents a significant challenge for hip surgeons. Preoperative diagnosis is not always easy to establish, making the intraoperative decision-making process crucial in deciding between one- and two-stage revision total hip arthroplasty (THA). Calprotectin is a promising point-of-care novel biomarker that has displayed high accuracy in detecting PJI. We aimed to evaluate the utility of intraoperative calprotectin lateral flow immunoassay (LFI) in THA patients with suspected chronic PJI. Methods. The study included 48 THAs in 48 patients with a clinical suspicion of PJI, but who did not meet European Bone and Joint Infection Society (EBJIS) PJI criteria preoperatively, out of 105 patients undergoing revision THA at our institution for possible PJI between November 2020 and December 2022. Intraoperatively, synovial fluid calprotectin was measured with LFI. Cases with calprotectin levels ≥ 50 mg/l were considered infected and treated with two-stage revision THA; in negative cases, one-stage revision was performed. At least five tissue cultures were obtained; the implants removed were sent for sonication. Results. Calprotectin was positive (≥ 50 mg/l) in 27 cases; out of these, 25 had positive tissue cultures and/or sonication. Calprotectin was negative in 21 cases. There was one false negative case, which had positive tissue cultures. Calprotectin showed an area under the curve of 0.917, sensitivity of 96.2%, specificity of 90.9%, positive predictive value of 92.6%, negative predictive value of 95.2%, positive likelihood ratio of 10.6, and negative likelihood ratio of 0.04. Overall, 45/48 patients were correctly diagnosed and treated by our algorithm, which included intraoperative calprotectin measurement. This yielded a 93.8% concordance with postoperatively assessed EBJIS criteria. Conclusion. Calprotectin can be a valuable tool in facilitating the intraoperative decision-making process for cases in which chronic PJI is suspected and diagnosis cannot be established preoperatively. Cite this article: Bone Joint J 2024;106-B(5 Supple B):118–124

Aims. This study aimed to investigate the clinical characteristics and outcomes associated with culture-negative limb osteomyelitis patients. Methods. A total of 1,047 limb osteomyelitis patients aged 18 years or older who underwent debridement and intraoperative culture at our clinic centre from 1 January 2011 to 31 December 2020 were included. Patient characteristics, infection eradication, and complications were analyzed between culture-negative and culture-positive cohorts. Results. Of these patients, 264 (25.2%) had negative cultures. Patients with a culture-negative compared with a culture-positive status were more likely to have the following characteristics: younger age (≤ 40 years) (113/264 (42.8%) vs 257/783 (32.8%); p = 0.004), a haematogenous aetiology (75/264 (28.4%) vs 150/783 (19.2%); p = 0.002), Cierny-Mader host A (79/264 (29.9%) vs 142/783 (18.1%); p < 0.001), antibiotic use before sampling (34/264 (12.9%) vs 41/783 (5.2%); p＜0.001), fewer taken samples (n＜3) (48/264 (18.2%) vs 60/783 (7.7%); p＜0.001), and less frequent presentation with a sinus (156/264 (59.1%) vs 665/783 (84.9%); p < 0.001). After initial treatments of first-debridement and antimicrobial, infection eradication was inferior in culture-positive osteomyelitis patients, with a 2.24-fold increase (odds ratio 2.24 (95% confidence interval 1.42 to 3.52)) in the redebridement rate following multivariate analysis. No statistically significant differences were found in long-term recurrence and complications within the two-year follow-up. Conclusion. We identified several factors being associated with the culture-negative result in osteomyelitis patients. In addition, the data also indicate that culture negativity is a positive prognostic factor in early infection eradication. These results constitute the basis of optimizing clinical management and patient consultations. Cite this article: Bone Joint J 2024;106-B(7):720–727