Aims. The aim of this study was to analyze the prevalence of culture-negative periprosthetic joint infections (PJIs) when adequate methods of culture are used, and to evaluate the outcome in patients who were treated with antibiotics for a culture-negative PJI compared with those in whom antibiotics were withheld. Methods. A multicentre observational study was undertaken: 1,553 acute and 1,556 chronic PJIs, diagnosed between 2013 and 2018, were retrospectively analyzed. Culture-negative PJIs were diagnosed according to the Muskuloskeletal Infection Society (MSIS), International Consensus Meeting (ICM), and European Bone and Joint Society (EBJIS) definitions. The primary outcome was recurrent infection, and the secondary outcome was removal of the prosthetic components for any indication, both during a follow-up period of two years. Results. None of the acute PJIs and 70 of the chronic PJIs (4.7%) were culture-negative; a total of 36 culture-negative PJIs (51%) were treated with antibiotics, particularly those with histological signs of infection. After two years of follow-up, no recurrent infections occurred in patients in whom antibiotics were withheld. The requirement for removal of the components for any indication during follow-up was not significantly different in those who received antibiotics compared with those in whom antibiotics were withheld (7.1% vs 2.9%; p = 0.431). Conclusion. When adequate methods of culture are used, the incidence of culture-negative PJIs is low. In patients with culture-negative PJI, antibiotic treatment can probably be withheld if there are no histological signs of infection. In all other patients, diagnostic efforts should be made to identify the causative microorganism by means of serology or molecular techniques. Cite this article: Bone Joint J 2022;104-B(1):183–188

Introduction

Infection rates following arthroplasty surgery are reported between 1–4%, with considerably higher rates in revision surgery. The associated costs of treating infected arthroplasty cases are over 4 times the cost of primary arthroplasties, with significantly worse functional and satisfaction outcomes. In addition, multiple antibiotic resistant bacteria are developing, so to reduce the infection rates and costs associated with arthroplasty surgery, new preventative methods are required. HINS-light is a novel blue light inactivation technology which kills bacteria through a photodynamic process, and is proven to have bactericidal activity against a wide range of species. The aim of this study was to investigate the efficacy of HINS-light for the inactivation of bacteria isolated from infected arthoplasty cases.

Aim

Propionibacterium acnes is a skin commensal colonizing the deeper structures of the pilous bulb. It is responsible for 5–10% of lower limb prosthetic joint infections (PJI) but accounts for as many as 50% of shoulder arthroplasty infections. P. acnes PJIs characteristically feature limited systemic inflammation, limited polymorphonuclear infiltration and clinical signs compatible with aseptic loosening. All current microbiological definitions of PJI require two or more identical commensal isolates to be recovered from the same procedure to diagnose PJI to increase specificity and rule out contamination. Whereas the antimicrobial susceptibility patterns of coagulase negative staphylococci are highly polymorphic and commonly allow the ready distinction of unrelated strains, P. acnes shows a highly stereotypical susceptibility profile and it is impossible to phenotypically assess the clonal relationship of isolates. In order to determine the clonal relationship of multiple P. acnes isolates recovered from arthroplasty revisions, we analyzed by multi-locus sequence typing (MLST) P. acnes isolates grown from PJI in a reference center for bone and joint infection.

Background. Data regarding the diagnostic value of ultrasound (US)-determined fluid film and joint aspiration prior to revision total hip arthroplasty (THA) for suspected periprosthetic joint infections (PJIs) is limited. This study aimed to analyse (1) the value of US-determined fluid film, (2) characterisation of the pre- and intraoperative microbiological spectrum and resistance patterns and (3) the concordance between preoperative synovial fluid and intraoperative culture results. Methods. We analysed 366 US-examinations from 340 patients prior to revision THA. Selected cases were categorized into clearly infected, non-infected and inconclusive, according to the International Consensus Meeting (ICM) 2018 Criteria. If US-determined fluid film was <1mm, no aspiration was performed based on our institutional standard protocol. Patients were grouped into no-aspiration (144/366;[39.3%]), dry-tap (21/366;[5.7%]) and a successful-tap (201/366;[54.9%]). The microbiological spectrum and antibiotic resistance patterns were determined and differences were compared between pre- and intraoperative results. Results. The absence of US-determined fluid film showed no correlation with the presence of hip PJI. Overall, 29.9% cases of the no-aspiration-group had a confirmed PJI. Discrepancies were found in 43.2% between successful taps and intraoperative cultures. The most prevalent microorganisms in preoperative synovial fluid were Staphylococcus epidermidis (20.9%), Staphylococcus aureus (20.9%) and Enterococcus faecalis (9.3%). The most prevalent microorganisms in intraoperative cultures were Staphylococcus epidermidis, Cutibacterium acnes and other coagulase-negative Staphylococci (14.2%). Additional microorganisms were identified in 43.8% intraoperatively. Staphylococcus aureus was more often detected preoperatively (20.9% vs. 5.8%;P=0.003), and Cutibacterium acnes intraoperatively (2.3% vs. 14.4%;P=0.01). There were no differences between the antibiotic resistance patterns of pre- and intraoperative concordant microorganisms. Conclusion. Absence of US-determined fluid film cannot rule out the presence of hip PJI. US-guided joint aspirations is a well-established technique. However, the preoperative analysis of synovial fluid shows high discrepancies especially in Cutibacterium acnes and other rare gram-positive microorganisms compared to intraoperative cultures

Aim. Culture-based conventional methods are still the gold standard to identify microorganisms in hip and knee PJIs diagnosis. However, such approach presents some limitations due to prior antimicrobial treatment or the presence of unusual and fastidious organisms. Molecular techniques, in particular specific real-time and broad-range polymerase chain reaction (PCR), are available for diagnostic use in a suspected PJI. However, limited data is available on their sensitivity and specificity. This study aimed to evaluate the performance of a rapid and simple Investigational Use Only (IUO) version of the BioFire® JI multiplex PCR panel when compared to traditional microbiological procedures. Method. Fifty-eight native synovial fluid samples were recovered from 49 patients (female n=26; male =23) who underwent one or multiple septic or aseptic revision arthroplasties of the hip (n=12) and knee (n=46). The JI panel methodology was used either on specimens freshly collected (n=6) or stored at −80°C in our Musculoskeletal Biobank (n=52). The JI panel performance was evaluated by comparison with culture reference methods. Patient's medical records were retrieved from our institutional arthroplasty registry as well as our prospectively maintained PJI infection database. Results. The JI panel identified additional microorganisms in 3/39 (7.7%) positive cases, and a different microorganism in 1/39 (2.6%) sample. Out of 9/58 (15.5%) culture negative samples, two (22%) were positively detected by the JI panel. In total 49/58 (84%) native synovial fluid specimens were positive by culture methods, versus 39/58 (81.2%) with the JI panel. Ten samples are currently under investigation for confirmatory results. Out of 39 positive detections with the JI panel, 35 (89.7%) were concordant with the identified microorganism (n=29 same species; n=6 same genus). The combined information from the JI panel results and clinical records revealed the existence of 6/58 (10.3%) PJIs’ cases which would have required a different antibiotic therapeutic approach. Conclusions. The work presented, provides additional value for the clinical use of the JI panel to the improvement of PJI management in terms of rapid and successful treatment decisions, patient outcome, and healthcare costs. This technique shows high sensitivity to detect PJIs specific microorganisms in both fresh as well frozen native synovial fluid samples, thus emphasizing its use for retrospective studies analysis

Aim. In severe cases of postoperative spinal implant infections (PSII) multiple revision surgeries may be needed. Little is known if changes of the microbiological spectrum and antibiotic resistance pattern occur between revision surgeries. Therefore, the aim of this study was to analyze the microbiological spectrum and antibiotic resistance pattern in patients with multiple revision surgeries for the treatment of PSII. Furthermore, changes of the microbiological spectrum, distribution of mono vs. polymicrobial infections, and changes of the antimicrobial resistance profile in persistent microorganisms were evaluated. Method. A retrospective analysis of a prospectively maintained single center spine infection database was performed with a minimum follow-up of 3 years. Between 01/2011 and 12/2018, 103 patients underwent 248 revision surgeries for the treatment of PSII. Overall, 20 patients (6 male/14 female) underwent 82 revisions for PSII (median 3; range 2–12). There were 55/82 (67.1%) procedures with a positive microbiological result. Microbiological analysis was performed on tissue and implant sonication fluid. Changes in microbial spectrum and antibiotic resistance pattern between surgeries were evaluated using Chi-Square and Fisher's exact test. Results. In total, 74 microorganisms (83.3% gram-positive; 10.8% gram-negative) were identified. The most common microorganisms were Staphylococcus epidermidis (18.9%) and Cutibacterium acnes (18.9%). All S. epidermidis identified were methicillin-resistant (MRSE). Overall, there were 15/55 (27.3%) polymicrobial infections. The microbiological spectrum changed in 57.1% (20/35) between the revision stages over the entire PSII period. In 42.9% (15/35) the microorganism persisted between the revision surgeries stages. Overall, changes of the antibiotic resistance pattern were seen in 17.4% (8/46) of the detected microorganisms comparing index revision and all subsequent re-revisions. Moreover, higher resistance rates were found for moxifloxacin and for ciprofloxacin at first re-revision surgery compared with index PSII revision. Resistances against vancomycin increased from 4.5% (1/23) at index PSII revision to 7.7% (2/26) at first re-revision surgery. Conclusions. Changes of the microbiological spectrum and the resistance pattern can occur in patients with severe PSII who require multiple revision surgeries. It is important to consider these findings in the antimicrobial treatment of PSII. The microbiological analysis of intraoperative tissue samples should be performed at every revision procedure for PSI

Purpose. Unexpected-positive-intraoperative-cultures (UPIC) in presumed aseptic revision-total-knee-arthroplasties (rTKA) are common, and the clinical significance is not entirely clear. In contrast, in some presumably septic rTKA, an identification of an underlying pathogen was not possible, so called unexpected-negative-intraoperative-cultures (UNIC). The purpose of this study was to evaluate alpha defensin (AD) levels in these patient populations. Methods. In this retrospective analysis of our prospectively maintained biobank, we evaluated synovial AD levels from 143 rTKAs. The 2018-Musculoskeletal Infection Society score (MSIS) was used to define our study groups. Overall, 20 rTKA with UPIC with a minimum of one positive intraoperative culture with MSIS 2-≥6 and 14 UNIC samples with MSIS≥6 were compared to 34 septic culture-positive samples (MSIS ≥6) and 75 aseptic culture-negative (MSIS 0–1) rTKAs. Moreover, we compared the performance of both AD-lateral-flow-assay (ADLF) and an enzyme-linked-immunosorbent-assay (ELISA) to test the presence of AD in native and centrifuged synovial fluid. Concentration of AD determined by ELISA and ADLF methods, as well as microbiological, and histopathological results, serum and synovial parameters along with demographic factors were considered. Results. AD was detected in 31/34 (91.2%) samples from the infected-group and in 14/14 (100%) samples in the UNIC group. All UPIC samples showed a negative AD result. Positive AD samples were highly (p<0.001) associated with culture positive and infection related histopathological results. Moreover, we found significantly (p=0.001) more high-virulent microorganisms 19/34 (55.9%) in the infected-group compared to the UPIC-group (0/20). Samples from the infected group with high virulent microorganisms 17/19 (89.5%) showed a positive AD. The presence of methicillin resistant Staphylococcus epidermis (MRSE) led to increased AD (p=0.003) levels when compared to those determined in samples positive for methicillin susceptible S. epidermdis (MSSE). ELISA and ADLF tests were positive with centrifuged (8/8) and native (8/8) synovial fluid. Conclusion. AD showed a solid diagnostic performance in infected and non-infected revisions, and it provided an additional value in the diagnostic of UPIC and UNIC associated to rTKAs. AD levels produced by patients with PJIs caused by high-virulent microorganisms and MRSE are significantly higher compared to those in patients with PJIs caused by either low-virulent or antibiotic susceptible microorganisms. Centrifugation of synovial fluid had no influence in the outcome of ADLF quantification. Keywords: Alpha-defensin, UPIC, UNIC, revision-knee-arthroplasty

Aim. Bone and joint infections (BJIs) are serious infections requiring early optimized antimicrobial therapy. BJIs can be polymicrobial or caused by fastidious bacteria, and the patient may have received antibiotics prior to sampling, which may decrease the sensitivity of culture-based diagnosis. Furthermore, culture-based diagnosis can take up to 14 days. Molecular approaches can be useful to overcome these concerns. The BioFire® system performs syndromic multiplex PCR in 1 hour, with only a few minutes of sample preparation. The BioFire® Joint Infection (JI) panel (BF-JI), recently FDA-cleared, detects both Gram-positive (n=15) and Gram-negative bacteria (n=14), Candida, and eight antibiotic resistance genes directly from synovial fluids. The aim of this study was to evaluate its performance in acute JIs in real-life conditions. Method. BF-JI was performed on synovial fluid from patients with clinical suspicion of acute JI, either septic arthritis or periprosthetic JI, in 6 French centers. The results of BF-JI were compared with the results of culture of synovial fluid and other concomitantly collected osteoarticular samples obtained in routine testing in the clinical microbiology laboratory. Results. From July 2021 to May 2022, 319 patients (including 10 children < 5y and 136 periprosthetic infections) had been included in the study. The BF-JI test was invalid for one patient (not retested). Among the 318 remaining patients, overall concordance with comparative microbiology methods was 88% (280/318): 131 samples were negative with both BF-JI and culture, and 149 samples were positive with the same microorganisms using complementary techniques. In 33 cases (10.4%), BF-JI was negative while culture was positive: 18 microorganisms were not targeted by BF-JI (including Staphylococcus epidermidis, n=10, and Cutibacterium acnes, n=2); 15 microorganisms targeted by BF-JI were obtained in culture but not by the molecular test (false-negative 4.7%). In 20 cases, BF-JI was positive while culture was not: 12 patients had received antibiotics before sampling, and 7 cases involved fragile and fastidious bacteria (Kingella kingae, n=5; Neisseria gonorrhoeae, n=2). In 6 cases, both BF-JI and culture were positive, but no yielding the same bacteria (polymicrobial specimens). Conclusions. In acute JIs, the BF-JI panel shows a good concordance with culture for the microorganisms targeted by the panel. Therefore, this molecular tool may have a place in microbiological diagnosis of acute JIs in order to confirm JI faster than culture. Moreover, it allows easy detection of difficult-to-culture bacteria. Acknowledgements. study was supported by bioMérieux, who provided all reagents

Aim. Treatment recommendations for periprosthetic joint infections (PJI) include surgical debridement, antibiotic therapy or staged revision. In surgical related foot and ankle infections (SR-FAI), implant removal will lead to instability. Debridement is difficult because the implant is outside the joint. Recommendations regarding PJI treatment can therefore not be extrapolated to the treatment of SR-FAI. Method. We searched PubMed for the etiology and treatment of SR-FAI, taken into account the time of occurrence, causative microorganisms and surgical treatment options. We integrated this knowledge into a treatment algorithm for SR-FAI. Results. Within the first 6 weeks after surgery, it is difficult to distinguish acute osteomyelitis from surgical site infection in which infection is limited to the soft tissue. The predominantly causative microorganism is Staphylococcus aureus. No debridement can be performed, because of the diffuse soft tissue inflammation and the absence of a joint space. If early SR- FAI is suspected without signs of systemic symptoms, fistula or abscess, empirical antibiotic treatment covering Staphylococcus aureus is recommended. If there is suspicion of ongoing SR-FAI after 2 weeks of empirical treatment, samples for culture after an antibiotic free window should be obtained to identify the causative microorganisms. If SR-FAI is confirmed, but there is no consolidation yet, targeted antibiotic treatment is given for 12 weeks without initial implant removal. In all other cases, debridement and samples for culture should be obtained after an antibiotic free window. Staged revision surgery will be performed if there is still a nonunion. Conclusions. Treatment algorithm regarding PJI cannot be extrapolated to the treatment of SR-FAI. Until now, no treatment guideline for SR-FAI is available. We have introduced a treatment algorithm for the treatment of SR-FAI. The guideline will be validated during the next 2 years

Aim. Fast and accurate identification of pathogens causing periprosthetic joint infections (PJI) is essential to initiate effective antimicrobial treatment. Culture-based approaches frequently yield false negative results, despite clear signs of infection. This may be due to the use of general growth media, which do not mimic the conditions at site of infection. Possible alternative approaches include DNA-based techniques, the use of in vivo-like media and isothermal microcalorimetry (ITC). We developed a synthetic synovial fluid (SSF) medium that closely resembles the in vivo microenvironment and allows to grow and study PJI pathogens in physiologically relevant conditions. In this study we investigated whether the use of ITC in combination with the SSF medium can improve accuracy and time to detection in the context of PJI. Methods. In this study, 120 synovial fluid samples were included, aspirated from patients with clinical signs of PJI. For these samples microbiology data (obtained in the clinical microbiology lab using standard procedures) and next generation sequencing (NGS) data, were available. The samples were incubated in the SSF medium at different oxygen levels (21% O. 2. , 3% O. 2. and 0% O. 2. ) for 10 days. Every 24h, the presence of growth was checked. From positive samples, cultures were purified on Columbia blood agar and identified using MALDI-TOF. In parallel, heat produced by metabolically active microorganisms present in the samples was measured using ITC (calScreener, Symcel), (96h at 37°C, in SSF, BHI and thioglycolate). From the resulting thermograms the ‘time to activity’ could be derived. The accuracy and time to detection were compared between the different detection methods. Results. So far, seven samples were investigated. Using conventional culture-based techniques only 14.3% of the samples resulted in positive cultures, whereas NGS indicated the presence of microorganisms in 57.1% of the samples (with 3/7 samples being polymicrobial). Strikingly, 100% of the samples resulted in positive cultures after incubation in the SSF medium, with time to detection varying from 1 to 9 days. MALDI-TOF revealed all samples to be polymicrobial after cultivation in SSF, identifying organisms not detected by conventional techniques or NGS. For the samples investigated so far, signals obtained with ITC were low, probably reflecting the low microbial load in the first set of samples. Conclusion. These initial results highlight the potential of the SSF medium as an alternative culture medium to detect microorganisms in PJI context. Further studies with additional samples are ongoing; in addition, the microcalorimetry workflow is being optimized

Aim. To date, no ultimate diagnostic gold standard for prosthetic joint infections (PJI) has been established. In recent years, next generation sequencing (NGS) has emerged as a promising new tool, especially in culture-negative samples. In this prospective study, we performed metagenomic analysis using 16S rRNA V3-V4 amplicon NGS in samples from patients with suspected PJI. Methods. A total of 257 (187 culture-negative (CN) and 70 culture-positive (CP)) prospectively collected tissues and sonication fluid from 32 patients (56 revisions) were included. 16S rRNA V3-V4 amplicons were sequenced using Illumina's MiSeq (California, USA) followed by bioinformatic analysis using nf-core/ampliseq pipeline. Results. We successfully sequenced 255 samples and detected a total of 105 microorganisms. These were mainly environmental microorganisms present in a small number of reads (≤100), indicating possible contamination. Pseudomonas spp. (non-aeruginosa species) was detected most frequently in 73% (187/255) of samples. The test showed limitations in species classification and identified microorganisms mainly at genus level. Significant differences in the number of reads were observed when comparing CN (≤100) and CP (≥1000) samples. In two CP, no bacteria were identified with sequencing, which is probably due to low bacterial load (1 CFU. Haemophilus spp. was detected with a significant number of reads (≥10000) in five samples from a single patient, in whom infection was considered likely according to EBJIS criteria, changing it to confirmed infection. Staphylococcus spp. was identified with ≥10000 reads in two CNs from an individual who was receiving antibiotic treatment at the time, had clinical signs of infection, and had a confirmed infection with S. lugdunensis one month earlier. Cutibacterium spp. with 36% (93/257) and Staphylococcus spp. with 34% (87/257) were detected with a minimal number of reads (≤100) in several CN, indicating possible contamination with normal skin microbiota. In one patient, Facklamia spp., an opportunistic pathogen, was detected in two samples by sequencing, but not by culture. Conclusions. We consider 16S rRNA V3-V4 amplicon sequencing to be a promising tool; however, further studies are needed to clarify uncertainties regarding the interpretation of the results in combination with other criteria. Using this method, we were able to successfully confirm infection in two patients whose microbiological results were initially negative, leading to a change from likely to confirmed infection in one case. The thresholds and interpretation of the results are currently unclear, therefore the method is being used experimentally rather than diagnostically at the time of writing

Aims. The aim of this study was to assess the incidence the microbiological spectrum and clinical outcome of hip and knee revision arthroplasties with unexpected-positive-intraoperative-cultures (UPIC) at a single center with minimum follow up of 2 years. Methods. We retrospectively analyzed our prospectively maintained institutional arthroplasty registry. Between 2011 and 2020 we performed presumably aseptic rTHA (n=939) and rTKA (n= 1,058). Clinical outcome, re-revision rates and causes as well as the microbiological spectrum were evaluated. Results. In total, 219/939 (23.3%) rTHA and 114/ 1,058 (10.8%) rTKA had a UPIC (p<0.001). Single positive intraoperative cultures were found in 173/219 (78.9%) in rTHA and 99/114 (86.8%) in rTKA, whereas 46/219 (21.0%) rTHA and 15/114 (13.2%) rTKA had positive results in ≥2 intraoperative cultures. A total of 390 microorganisms were found among the 333 cases. Staphylococcus epidermidis 30.9%, CoNS (21.9%), Cutibacterium acnes 21.1%, and Bacillus spp. 7.3% were the most common microorganisms. Overall, detected microorganisms showed high sensitivity to daptomycin (96.6%), vancomycin (97.3%) and linezolid (98.0%). After a minimum follow up of 2 years (rTHA 1,470 (735; 3,738) days; rTKA 1,474 (749; 4,055) days). During the 2-year follow-up, 8 patients died and 5 were lost to follow-up. There were 54/219 (24.7%) re-revision in rTHa and 20/114 (17.5%) in rTKA. Overall, there were 23 (10.5%) septic re-rTHA and 9 (7.9%) septic re-rTKA as well as 31 (14.2%) aseptic re-rTHA and 11 (9.6%) aseptic re-rTKA. Patients with previous septic revisions bevor UPIC procedure showed a significant higher risk for septic re-revision (p<0.05). Moreover, there were less septic re-revisions after single culture positive UPIC (rTHA: 16/173 (9.2%); rTKA 6/99 (6.1%)) compared to ≥2 positive intraoperative cultures UPIC (rTHA: 7/46 (15.2%); rTKA 3/15 (20.0%)). The most common reason for re-revision in the rTHA-group was aseptic loosening of the cup (34.2%) or of the stem (23.3%), dislocation (18.3%) and periprosthetic-fractures (7.8%). In the rTKA-group it was aseptic loosening (40.4%), instability (24.6%) and secondary patella resurfacing (7.9%). There was a higher septic re-revision rate in consecutive revisions than in planned revisions 17.3% vs. 8.5% in the rTHA-group and 14.3% vs. 7.5% in the rTKA-group, p<0.001. Conclusion. UPICs are common in rTJA. The rate was higher in hips which may partly explained by the easier pre op joint aspiration in the knee. UPIC may lead to an increase in subsequent re-revisions

Aim. Unexpected negative-cultures (UNC) are a common diagnostic problem in periprosthetic joint infection (PJI) of the hip and knee when using culture-based methods. A novel molecular approach (MC)1 based on the identification of the vast majority of bacterial species in a single assay using species-specific bacterial interspacing region length polymorphisms and phylum-specific 16S rDNA sequence polymorphisms has demonstrated clinical utility in PJI diagnostics (1). In addition, MC provides an estimate of the leukocyte concentration in the specimen analysed. The aim of this retrospective, blinded study was to evaluate the performance of MC in identifying the microbiological content and determining the leukocyte count in synovial fluid (SF) collected from hip and knee revision arthroplasty cases with UNC. It was also assessed whether antibiotic treatment would have been changed if the result from MC had been known. Method. A total of 89 SF samples from 70 patients (43 female; 27 male) who underwent revision arthroplasty (14 hip; 75 knee) were included. Using European and Bone Joint Infection Society (EBJIS) criteria, 82 cases were classified as infected (77 UNC and 5 septic culture-positive controls), five as non-infected (aseptic culture-negative controls), and two as likely infected, but infected by clinical observation. MC was performed and evaluated together with SF parameters. Antibiotic treatment, clinical outcome, patient demographics and surgical details were analysed. Results. Overall, 29.1% (23/79) of UNC had a positive yield by MC, of which 2/23 (8.7%) had two microorganisms detected simultaneously. Of the 25 microorganisms identified by MC, 12/25 (48%) were clinically relevant after re-evaluation of the patients’ microbiological history. The microorganisms detected were 5/25 (20%) Streptococcus pneumoniae/mitis, 4/25 (16%) Staphylococcus epidermidis, 3/25 (12%) Cutibacterium acnes, 3/25 (12%) Streptococcus agalactiae, 2/25 (8%) Streptococcus bovis, 2/25 (8%) Staphylococcus aureus, and 2/25 (8%) Haemophilus parainfluenzae. The prevalence of Enterococcus faecalis, Bacteroides fragillis, Staphylococcus lugdunensis, Corynebacterium striatum among all MC results was 1/25 (4%) each species. In total, 13/23 (56%) cases were associated with patients receiving antibiotic therapy at the time of SF collection. The yield for leukocyte counts provided the molecular technique was consistently much higher in the UNC and clearly septic groups than in the clearly aseptic group. Overall, 20/61 (32.8%) patients with UNC could have been managed differently and more accurately after MC assessment. Conclusions. MC shows clinical value in the diagnosis and management of PJI with UNC. The included leukocyte count shows promising results. Acknowledgments. This work was partially funded by Inbiome

Aim. Diagnosing low-grade periprosthetic joint infections (PJI) can be very challenging due to low-virulent microorganisms capable of forming biofilm. Clinical signs can be subtle and may be similar to those of aseptic failure. To minimize morbidity and mortality and to preserve quality of life, accurate diagnosis is essential. The aim of this study was to assess the performance of various diagnostic tests in diagnosing low-grade PJI. Methods. Patients undergoing revision surgery after total hip and knee arthroplasty were included in this retrospective cohort study. A standardized diagnostic workup was performed using the components of the 2021 European Bone and Joint Infection Society (EBJIS) definition of PJI. For statistical analyses, the respective test was excluded from the infection definition to eliminate incorporation bias. Receiver-operating-characteristic curves were used to calculate the diagnostic performance of each test, and their area-under-the-curves (AUC) were compared using the z-test. Results. 422 patients undergoing revision surgery after total hip and knee arthroplasty were included in this study. 208 cases (49.3%) were diagnosed as septic. Of those, 60 infections (28.8%) were defined as low-grade PJI (symptoms >4 weeks and caused by low-virulent microorganisms (e. g. coagulase-negative staphylococci, Cutibacterium spp., enterococci and Actinomyces)). Performances of the different test methods are listed in Table 1. Synovial fluid (SF) - WBC (white blood cell count) >3000G/L (0.902), SF - %PMN (percentage of polymorphonuclear neutrophils) > 65% (0.959), histology (0.948), and frozen section (0.925) showed the best AUCs. Conclusion. The confirmatory criteria according to the EBJIS definition showed almost ideal performances in ruling-in PJI (>99% specificity). Histology and synovial fluid cell count (SF-WBC and SF-%PMN) showed excellent accuracies for diagnosing low-grade PJI. However, a reduced immune reaction in these cases may necessitate lower cut-off values. Intraoperative frozen section may be valuable in cases with inconclusive preoperative diagnosis. For any tables or figures, please contact the authors directly

Aim. Our aim was to evaluate the prevalence and impact of unexpected intraoperative cultures on the outcome of total presumed aseptic knee and hip revision surgery. Method. Data regarding patients prospectively recruited in our center, who had undergone elective complete hip and knee revision surgery from January 2003 to July 2017 with a preoperative diagnosis of aseptic loosening was retrospectively reviewed. Partial revisions and patients with follow up below 60 months were excluded from the study. The protocol of revision included at least 3 intraoperative cultures. Failure was defined as the need for re-revision due to any-cause at 5 years and/or the need for antibiotic suppressive therapy. Results. A total of 608 cases were initially included in the study, 53 patients were excluded. 123 hip and 432 knee revision surgeries were included. 420 cases (75.7%) had all cultures negative, 114 (20.5%) a single positive culture or two of different microorganisms and 21 (3.8%) had at least 2 positive cultures for the same microorganism. Early failure was found in 4.8% (1/21) of the patients with missed low grade infection. The presence of positive cultures during total exchange was not associated with a higher failure rate than in those with negative cultures (44 of 420, 10.5%). In contrast, patients revised before 24 months had a significant higher rate of re-revision, 18% (15/83) vs. 8.4%. Conclusions. Total hip and knee revisions with unexpected positive cultures were not significantly associated with a higher re-revision risk at 5 years of follow-up. Representing an overall good prognosis. However, revision surgeries performed within the first 24 months have a higher rate of failure

Aim. To evaluate the bacterial counts of sonicatied implants in patients with osteoarticular infections. Various studies have demostrated the usefulness of sonication of retrieved implants in order to provide an accurate microbiological diagnosis. Although cutoff values for original sonicate counts have been established, the use of centrifugation may influence these values. Method. A retrospective, single-center study, including sonication fluid samples from implants removed between January 2011 and October 2023, was performed. Patients were diagnosed with implant-associated infection based on the criteria available at the time of diagnosis. Osteoarticular implants were sonicated following the protocol described by Esteban et al. Sonicated fluid was centrifuged for 20 minutes at 3000 x g, and the sediment was resuspended in 5 mL of phosphate buffer solution. Ten µl of the sample were streaked onto each medium for quantitative culture. Bacterial counts exceeding 100,000 CFU/mL were considered as 100,000 CFU/mL for statistical analysis. Results. The study included 457 sonication fluid samples. Of these, 316 samples were from patients with prosthetic joint infection (PJI), with 26.3 % diagnosed with acute PJI and 73.7 % with chronic PJI. Additionally, 141 samples were from patients with osteosynthesis infection. The median CFU/ml in the sonication fluid was 40,000 CFU/mL (IQR 1,000 CFU/mL-100,000 CFU/mL). No statistically significant difference was observed between the different types of implants (prosthesis vs. osteosynthesis, p=0.218). A trend of higher counts was noted for acute PJI compared to chronic PJI (р=0.052). Most infections were monomicrobial, but 16.2% were polymicrobial. Statistically significant higher bacterial counts were observed in polymicrobial infections compared to monomicrobial infections (р<0.005). Among monomicrobial infections, no differences were found between Gram-negative and Gram-positive microorganisms (р=0.416). No differences were also found between joints (knee vs. hip) (p=0.353). Conclusions. Significant variability was observed in the number of colonies detected in all samples, regardless of the type of implant, the number of microorganisms or the species identified. Higher counts were detected in polymicrobial infections, and a trend was also noted for higher counts in acute infections

Aim. This study aimed to evaluate the impact of intraoperative direct sonication on the yield of traditional culture and the time to positivity (TTP) of cultures obtained for periprosthetic joint infection (PJI), thereby assessing its potential to improve diagnostic efficiency and reduce contamination risk. Method. A prospective cohort study was conducted at a tertiary care center, involving 190 patients undergoing revision surgery for PJI from August 2021 to January 2024. Patients were included based on the 2018 International Consensus Meeting definition of PJI. The study utilized a novel sonication protocol, which involved direct intraoperative sonication of the implant and tissue, followed by incubation in a BACT/ALERT 3D system. The primary outcomes measured were the number and percentage of positive culture samples, identified microorganisms, and the TTP of each culture. Statistical analysis was performed using R software, with various tests applied to assess the significance of findings. Results. The study included 510 positive cultures from 190 patients, demonstrating that sonication significantly improved the positivity rate for both tissue and prosthesis specimens (p < 0.05). The median TTP for all samples was 3.13 days, with sonicated samples showing a significantly shorter TTP compared to non-sonicated samples (p < 0.05). Specifically, the shortest median TTP was observed in prosthesis post-sonication samples. Furthermore, the study found that Gram-positive organisms had a shorter TTP than gram-negative organisms, and specific microorganisms like Staphylococcus aureus and MRSE showed the fastest TTP. The analysis also revealed higher positivity rates in chronic PJIs compared to acute PJIs for sonicated tissue samples. Conclusions. The study demonstrates that intraoperative direct sonication combined with the BACT/ALERT 3D system can significantly enhance the diagnostic yield of cultures and reduce the TTP for common PJI pathogens. This novel technique not only improves pathogen detection, facilitating the tailoring of antibiotic therapy, but also potentially reduces the risk of contamination associated with sonication. These findings suggest that direct intraoperative sonication could be a valuable addition to the current diagnostic protocols for PJI, contributing to more effective management and treatment of this complex condition. Further research is necessary to explore the clinical significance of TTP and its correlation with patient outcomes in PJI

Aim. The purpose of this study is to analyze the demographic and microbiological variables of acute ankle infections posterior to ankle osteosynthesis and to determine the different characteristics of patients withE. cloacae infection. Method. A multicenter retrospective observational study (4 national hospitals) of acute post osteosynthesis infections of ankle fracture operated between 2015 and 2018 was implemented. The demographic and microbiological variables relating to the surgical intervention and the antibiotic treatment performed were collected. A descriptive assessment of all the variables and a univariate comparison between patients with E. cloacae infection and patients with alternative microorganism infections were performed. The SPSS v25 program for Windows was the choice for statistical analysis. Results. 71 Patients with an average age of 57 years were included, the majority being males (55%). 31% of patients were diabetic, 27% had vascular pathology, and 18.3% had a BMI greater than 35. Trimalleolar fracture was the most common in our study being 52%. 26.8% were open fractures. The microorganisms isolated were: 25% S. aureus, 22.5% E. cloacae and 22.5% polymicrobial. Accounting for polymicrobial infections, the presence of E. cloacae rises to 32%. In the univariate analysis, only significant differences were found in age (patients with E. cloacae infection were older) and the use of VAC therapy. Conclusions. In our series, higher percentages of E. cloacae infection were observed than those described in the literature. There are statistically significant differences in the variables of age and need for VAC therapy. The high incidence of E. cloacae infections suggests the vital importance of adapting antibiotic prophylaxis, ensuring the coverage of this microorganism

Aim. Prompt recognition and identification of the causative microorganism in acute septic arthritis of native and prosthetic joints is vital to increase the chances of successful treatment. The aim of this study was to independently assess the diagnostic accuracy of the multiplex BIOFIRE® Joint Infection (JI) Panel (investigational use only) in synovial fluid for rapid diagnosis. Method. Synovial fluid samples were prospectively collected at the University Medical Center Groningen from patients who had a clinical suspicion of a native septic arthritis, early acute (post-operative, within 3 months after arthroplasty) periprosthetic joint infection (PJI) or late acute (hematogenous) PJI. JI Panel results were compared to culture-based methods as reference standard. Results. A total of 45 samples were analyzed. The BIOFIRE JI Panel showed a high specificity (100%, 95% CI 73 – 100) and positive predictive value (100%, 95% CI 79 – 100) in all patient categories. Sensitivity and negative predictive value were 83% (95% CI 36 – 99) and 88% (95% CI 47 – 99) respectively for patients with a clinical suspicion of native septic arthritis (n=12), 77% (95% CI: 46 – 94) and 63% (95% CI: 26 – 90) for patients with a clinical suspicion of a late acute PJI (n=14), and 27% (95% CI 7 – 61) and 27% (95% CI: 7 – 61) for patients with a clinical suspicion of an early acute PJI (n=19). Conclusions. The results of this pilot study indicate a clear clinical benefit of the BIOFIRE JI Panel in patients with a suspected native septic arthritis and late acute (hematogenous) PJI, but a low clinical benefit in patients with an early acute (post-operative) PJI due to the absence of low-grade microorganisms in the panel

Aim. We prospectively evaluated four different microbiological tools for diagnostics of prosthetic joint infections (PJI), and assessed their impact on the categorization of infection according to EBJIS guidelines. We compared culture, in-house real-time mPCR for S. aureus, S. lugdunensis, S. hominis, S. epidermidis, S. capitis, S. haemolyticus, C. acnes (mPCR), broad-spectrum PCR (Molzym) with 16S rRNA V3-V4 amplicon Sanger sequencing (16S PCR), and 16S rRNA V3-V4 amplicon next-generation sequencing (16S NGS) on MiSeq (Ilumina). Methods. A total of 341 samples (sonication fluid, tissue biopsy, synovial fluid) were collected from 32 patients with suspected PJI who underwent 56 revision surgeries at the Orthopaedic Centre University Hospital Ljubljana, between 2022 and 2024. Samples were processed using standard protocols for routine culture, followed by DNA isolation using the MagnaPure24 (Roche). All samples were tested with mPCR, and an additional ≥4 samples from each revision (244 in total) were subjected to further metagenomic analysis. Culture results were considered positive if the same microorganism was detected in ≥2 samples, ≥50 CFU/ml were present in the sonication fluid, or ≥1 sample was positive for a more virulent microorganism or if the patient had received antibiotic treatment. Results. Each tool demonstrated high sensitivity for correct EBJIS categorization (100% culture and 16S NGS, 96.88% mPCR and 16S PCR). The highest specificity was observed with mPCR and 16S PCR (87.5%), while culture (79.17%) and NGS (37.5%) showed lower specificity. In 27% (15/56) of revisions, all microbiological tests were negative, although infection was confirmed with histology in one case, and four cases were classified as infection-likely based on clinical signs. In 20% (11/56) of cases, all microbiological tests were positive; in three cases a combination of other EBJIS criteria (without microbiology) categorized the episodes as infection-likely and one as infection-unlikely, emphasizing the importance of microbiological tests in diagnostic criteria. In 43% (24/56) of revisions categorized as infection-unlikely using a combination of other EBJIS criteria, five had positive culture, and three had positive mPCR and 16S PCR. Fifteen (62%) had positive 16S NGS, 12 due to a low number of reads, which may indicate low-grade infection or possible contamination. Conclusion. To date, no test can be established as the ultimate gold standard. The lack of interpretation criteria can result in low specificity of some methods, as the threshold is difficult to determine. A multidisciplinary approach with combination of microbiological tools is still considered the most efficient