AimPeriprosthetic joint infection (PJI) is one of the most devastating complications after joint replacement. It is associated with high morbidity and economic burden when misdiagnosed as an aseptic failure. Among all cases of PJI, up to 25% could yield negative cultures. Conversely, among cases of aseptic failures, up to 30% may actually be undiagnosed PJIs. In PJIs microbiological diagnosis is a key step for successful treatment. Sonication of the removed prosthesis is more sensitive than conventional periprosthetic-tissue culture, especially in patients who received antimicrobial therapy before surgery. This study aimed to compare the diagnostic value of classic sonication fluid cultures (SF-C) and sonication fluid incubation in blood culture bottle (SF-BCB).MethodBetween 2016 and 2018 we analysed 160 revision procedures of joint arthroplasties. For each procedure, at least 5 microbiological and multiple histopathological samples were harvested, and explant sonication was performed which was further analysed by SF-C and SF-BCB. For SF-C classical cultivation of sonication fluid was performed. While for SF-BCB, 10 mL of sonication fluid was inoculated into aerobic and anaerobic lytic blood culture bottles. The definite diagnosis of PJI was based on the EBJIS definition.ResultsAmong 160 revisions, 59 PJIs were identified, 15 patients were treated with the debridement and implant retention, 7 patients with the one-stage and 35 with the two-stage exchange, remaining 2 were partial revisions. The sensitivity of SF-C and SF-BCB were 81.5% and 94.9%, respectively. The mismatch of microbe identification was observed in 5 cases. We observed positive SF-C while negative SF-BCB in 4 cases, among them having 2 positive histology. While 12 patients have negative SF-C and positive SF-BCB, among them 3 have positive and 6 negative histology. Among these 12 patients, typical low-grade microbes were identified in 9 cases (5 cases of C. acnes, 3 cases of S. epidermidis, and 1 case of S. capitis).ConclusionsThe weakest point in all PJI diagnostic criteria is their sensitivity. SF-BCB demonstrates higher sensitivity in diagnosing PJI compared to SF-C. Therefore, it appears prudent to incorporate SF-BCB into the diagnostic protocol for all patients exhibiting either low-grade PJI symptoms or experiencing undiagnosed, presumably aseptic failures, where the likelihood of misdiagnosing infection is greatest.

Introduction: The BIOFIRE Joint Infection (JI) Panel is a diagnostic tool that uses multiplex-PCR testing to detect microorganisms in synovial fluid specimens from patients suspected of having septic arthritis (SA) on native joints or prosthetic joint infections (PJIs). Methods: A study was conducted across 34 clinical sites in 19 European and Middle Eastern countries from March 2021 to June 2022 to assess the effectiveness of the BIOFIRE JI Panel. Results: A total of 1527 samples were collected from patients suspected of SA or PJI, with an overall agreement of 88.4 % and 85 % respectively between the JI Panel and synovial fluid cultures (SFCs). The JI Panel detected more positive samples and microorganisms than SFC, with a notable difference on Staphylococcus aureus, Streptococcus species, Enterococcus faecalis, Kingella kingae, Neisseria gonorrhoeae, and anaerobic bacteria. The study found that the BIOFIRE JI Panel has a high utility in the real-world clinical setting for suspected SA and PJI, providing diagnostic results in approximately 1 h. The user experience was positive, implying a potential benefit of rapidity of results' turnover in optimising patient management strategies. Conclusion: The study suggests that the BIOFIRE JI Panel could potentially optimise patient management and antimicrobial therapy, thus highlighting its importance in the clinical setting.

Prosthetic joint infections (PJIs) are commonly diagnosed via culture-based methods, which may miss hard-togrow pathogens. This study contrasts amplicon metagenomic sequencing (16S AS) with traditional culture techniques for enhanced clinical decision-making. We analyzed sonicate fluid from 27 patients undergoing revision arthroplasty using both methods, emphasizing the distinction between contaminants and true positives. Our findings show moderate agreement between the two methods, with a Cohen's kappa of 0.490, varying across bacterial genera (Cohen's kappa -0.059 to 1). The sensitivity of 16S AS compared to culture was 81% (95% CI, 68% to 94%). Sequencing revealed greater microbial diversity, including anaerobic genera like Anaerococcus and Citrobacter. Interestingly, several culture-negative PJI samples showed diverse bacteria via 16S AS. Despite rigorous controls and algorithms to eliminate contaminants, confirming bacteria presence with 16S AS remains a challenge. This highlights the need for improved PJI diagnostic methods, while also pointing out the limitations of next-generation sequencing (NGS) as a clinical diagnostic tool.

Aim: To evaluate the accuracy of two PCR-based techniques for detecting SARS-CoV-2 variant Alpha (B.1.1.7). Materials & methods: A multicenter prospective cohort with 1137 positive specimens from Slovenia was studied. A mutation-based assay (rTEST-COVID-19 qPCR B.1.1.7 assay) and amplification curve pattern analysis of the Allplex SARS-CoV-2 assay were compared with whole-genome sequencing. Results: SARS-CoV-2 variant Alpha was detected in 155 samples (13.6%). Sensitivity and specificity were 98.1 and 98.0%, respectively, for the rTEST-COVID-19 qPCR B.1.1.7 assay and 97.4 and 97.5%, respectively, for amplification curve pattern analysis. Conclusion: The good analytical performance of both methods was confirmed for the preliminary identification of SARS-CoV-2 variant Alpha. This cost-effective principle for screening SARS-CoV-2 populations is also applicable to other emerging variants and may help to conserve some whole-genome sequencing resources.

ObjectivesBlood culture bottles (BCBs) are commonly used for the diagnosis of infections associated with orthopedic devices. Although Cutibacterium acnes is an important pathogen in orthopedics, relatively little is known about its growth characteristics in BCBs. This prompted us to analyze the influence of bacterial genotype and clinical significance on time-to-detection (TTD) in BCBs.MethodsWe reviewed 59 cases of orthopedic device-related infections in which at least one intraoperative specimen yielded a pure C. acnes culture from anaerobic BCBs (BD Bactec Lytic/10 Anaerobic/F; Lytic-Ana) and/or solid media. A strain was considered infectant if the same genotype was present in two or more intraoperative samples. From these cases, we isolated a total of 72 unique C. acnes strains belonging to four multilocus sequence type clonal complexes (CCs): CC18, CC28, CC36 and CC53. Growth rate and TTD in Lytic-Ana BCB were studied under experimental conditions (inoculation of standard inoculum) and in clinical samples (inoculation of periprosthetic tissue samples).ResultsMedian TTD values were shorter for CC53 compared to other CCs under experimental conditions (69 vs. 103 h; p < 0.001) and from clinical specimens (70 vs. 200 h; p = 0.02). Infectant strains had a shorter median TTD compared to contaminant strains in a clinical situation, while the difference was not observed under experimental conditions.ConclusionsThe detection dynamics of C. acnes in Lytic-Ana BCBs were associated with genotype. Thus, TTD not only reflects the bacterial load in clinical samples, but may also reflect the intrinsic properties of the clonal complex of C. acnes.

Electrochemotherapy combined with peritumoral interleukin-12 (IL-12) gene electrotransfer was used for treatment of mast cell tumours in 18 client-owned dogs. Local tumour control, recurrence rate, as well as safety of combined therapy were evaluated. One month after the therapy, no side effects were recorded and good local tumour control was observed with high complete responses rate which even increased during the observation period to 72%. IL-12 gene electrotransfer resulted in 78% of patients with detectable serum IFN-γ and/or IL-12 levels. In the treated tumours vascular changes as well as minimal T-lymphocytes infiltration was observed. After 1 week, the plasmid DNA was not detected intra- or peritumorally and no horizontal gene transfer was observed. In summary, our study demonstrates high antitumour efficacy of electrochemotherapy combined with IL-12 electrotransfer, which also prevented recurrences or distant metastases, as well as its safety and feasibility in treatment of canine mast cell tumours.

Background and purpose - The correct diagnosis of prosthetic joint infection (PJI) can be difficult because bacteria form a biofilm on the surface of the implant. The sensitivity of culture from sonication fluid is better than that from periprosthetic tissue, but no comparison studies using molecular methods on a large scale have been performed. We assessed whether periprosthetic tissue or sonication fluid should be used for molecular analysis. Patients and methods - Implant and tissue samples were retrieved from 87 patients who underwent revision operation of total knee or total hip arthroplasty. Both sample types were analyzed using broad-range (BR-) PCR targeting the 16S rRNA gene. The results were evaluated based on the definition of periprosthetic joint infection from the Workgroup of the Musculoskeletal Infection Society. Results - PJI was diagnosed in 29 patients, whereas aseptic failure was diagnosed in 58 patients. Analysis of sonication fluid using BR-PCR detected bacteria in 27 patients, whereas analysis of periprosthetic tissue by BR-PCR detected bacteria in 22 patients. In 6 of 7 patients in whom BR-PCR analysis of periprosthetic tissue was negative, low-virulence bacteria were present. The sensitivity and specificity values for periprosthetic tissue were 76% and 93%, respectively, and the sensitivity and specificity values for sonication fluid were 95% and 97%. Interpretation - Our results suggest that sonication fluid may be a more appropriate sample than periprosthetic tissue for BR-PCR analysis in patients with PJI. However, further investigation is required to improve detection of bacteria in patients with so-called aseptic failure.

Abstract Electrochemotherapy is now well established ablative local tumor treatment for veterinary as well as human cancer. Electrochemotherapy combines the use of permeabilizing electric pulses applied directly at the tumor site with the injection of chemotherapeutic drugs, cisplatin or bleomycin. Increased membrane permeability enables enhanced entry of chemotherapeutic drug into the cells and resulted in higher tumor cell kill. Up to 80% complete responses are obtained after single electrochemotherapy treatment of cutaneous tumors of different histology (Yarmush et al. Annu Rev Biomed Eng. 2014; 16:295-320). To add a systemic component to this very effective local treatment, in preclinical studies it was combined with several different immune therapies, including gene therapy with plasmid DNA encoding interleukin-12 (IL-12) (Radiol Oncol. 2012;46:302-11). In our on-going clinical research, the effect of IL-12 gene therapy combined with electrochemotherapy was evaluated in 18 client- owned dogs with mastocytomas. Written consent for participation was obtained from the owners and the study was approved by the competent authorities (Veterinary administration and Administration for biotechnology of the Ministry of agriculture and environment; Republic Ethical Committee for the experiments involving animals). Electrochemotherapy using intratumoral injection of bleomycin and application of electric pulses was preformed prior to gene therapy. Plasmid encoding IL-12 was injected peritumorally into the skin and immediately thereafter electric pulses were applied. Local response of tumors was evaluated by measurements of tumors’ size. Systemic response was assessed by determination of IL-12 and IFN-γ in patients’ sera. Safety of gene therapy was evaluated by qRT-PCR measurement for the presence of plasmid DNA at the site of application and possible horizontal gene transfer by in vitro transformation of plasmid DNA encoding IL-12 into the bacteria isolated from the dog's (patient’s) skin. One month after the therapy, 11 dogs (61%) had a complete response, 4 (22%) partial response, and 3 (16%) stable disease. At the end of observation period (median 25 months) 16 dogs (89%) had complete response and 2 had to be euthanized due to the progression of disease. In all dogs, except two, serum IFN-γ or IL-12 levels were detected, without systemic toxicity. At the site of DNA plasmid application, except for one patient, the plasmid was not detected at 1 week post-treatment. No horizontal gene transfer was detected; plasmid was not detected in any of the residential bacteria and in vitro transformation of plasmid into isolated strains was negative. Collectively, the results of our on-going clinical trial demonstrate that IL-12 gene therapy combined with electrochemotherapy is safe, feasible and effective treatment for canine mastocytoma. Citation Format: Maja Cemazar, Jerneja Ambrozic Avgustin, Gregor Sersa, Darja Pavlin, Ana Krhac Levacic, Natasa Tesic, Mitja Rak, Ursa Lampreht, Natasa Tozon. Interleukin-12 gene therapy combined with local ablative technique electrochemotherapy for treatment of canine mastocytoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 292. doi:10.1158/1538-7445.AM2015-292

Overthe past half-century, there have been many advances in the design, construction, and implantation of joint prostheses, resulting in a high percentage of successful long-term outcomes. One of the most common concerns of both patients and physicians is the problem of joint replacements becoming loose over time. Causes of failure include infections, aseptic loosening, dislocations, and fracture of the prosthesis or bone. Multidisciplinary research team studies are needed for an improvement in understanding in pathophysiological mechanisms of joint implant loosening and failure, which is the key point to improve implant survival and to minimize revisions. Index Terms — diagnosis, infection, loosening, prosthetic joints

Polymerase chain reaction (PCR) has enabled enormous progress in the field of molecular biology in the last twenty-five years. It became popular due to its high sensitivity and specificity and was introduced to numerous scientific fields. Today many different variants of PCR exist, with real-time PCR being the most common. This method is being successfully used at our institution for a variety of different applications, among which gene expression analyzes and development of protocols for clinical diagnostics currently dominate. But the extreme flexibility and customizability of real-time PCR, coupled with our team’s expertise, ensures efficient application of this technique to various new research projects. Index Terms — real-time PCR, SYBR Green, TaqMan, quantification, gene expression

PURPOSE:Prosthetic joint infection (PJI) is a devastating complication of total joint arthroplasty. No single laboratory test has perfect sensitivity and specificity; however, culture of periprosthetic tissue is the standard method for PJI diagnosis. Interpretation of positive culture results in PJI diagnostics can be difficult due to the possibility of contamination with microorganisms originating from skin micro flora. Criteria have been established to aid in distinguishing pathogen from contaminant for culture results. A similar criterion has not however been established for polymerase chain reaction (PCR) analysis, which is in part responsible for confusion about the reliability of PCR for PJI diagnostics. The aim of our study was to establish a criterion for interpretation of broad range (BR) PCR results in PJI diagnostics.METHODS:Samples of periprosthetic tissue were retrieved from 100 patients with joint prosthesis failure and analysed with BR-PCR. The results of BR-PCR were evaluated based on the number of samples of periprosthetic tissue with the same bacterial species.RESULTS:The sensitivity (87.5%) of BR-PCR was highest if the same species was present in at least one sample, although this criterion also resulted in the lowest specificity (92.1%). The sensitivity decreased (83.2%), although without a statistically significant difference, if the same species was present in two or more samples but, at the same time, specificity increased (100%), with a statistically significant difference.CONCLUSIONS:For diagnostics of PJI with BR-PCR the criterion of the same bacterial species in at least two specimens of periprosthetic tissue from the same patient should be used for interpretation of positive results.

Diagnosis of prosthetic joint infection with culture technique can be problematic since the causative agent(s) are not possible to cultivate in all cases. Molecular methods had been evaluated in many studies but their inclusion in routine diagnostics is still controversial. The purpose of our prospective study was to compare the diagnostic accuracy of broad-range (BR)-PCR and culture technique. Intraoperative samples of periprosthetic tissue were retrieved in 67 patients undergoing revision arthroplasty. Samples were analyzed with culture technique, immunohistochemistry and BR 16S rRNA gene PCR. Bacteria in PCR-positive samples were identified using two different methods: direct sequencing of PCR products and specific TaqMan assays. In 63 cases, full concordance was found between BR-PCR and culture technique. Specific TaqMan assays failed to identify bacteria in four culture- and BR-PCR-positive cases and therefore had a lower sensitivity in comparison with BR-PCR. Molecular methods detected bacteria with the same accuracy as culture; however, identification of bacteria was inferior to culture. Further development of species-recognition techniques is required to improve identification of causative microorganisms.