Aims

This aim of this study was to analyze the detection rate of rare pathogens in bone and joint infections (BJIs) using metagenomic next-generation sequencing (mNGS), and the impact of mNGS on clinical diagnosis and treatment.

Intraoperative histology has a high specificity and sensitivity to identify prosthetic joint infection. However, the usefulness of this technique according to the type of microorganism isolated in the periprosthetic tissue has not previously been studied. Frozen sections and cultures from periprosthetic tissue of 38 revision arthroplasties performed due to prosthetic joint infection were retrospectively reviewed. Frozen sections were evaluated according to Mirras’ criteria (adapted by Feldman). Culture was considered positive when the same microorganism was isolated in at least 2 samples or the presence of pus around the prosthesis. Coagulase-negative staphylococci (CNS) was the aetiology in 13 cases, Gram-negative bacilli in 8, S. aureus in 7, Candida sp and Peptococcus sp in 2 and Enterococcus sp, S.pneumoniae and in 1 case each one. No microorganism was isolated in 4 cases. Frozen sections revealed more than 5 neuthrophils per high power field (forty times) in at least five fields in all cases except in 2 out of 13 caused by CNS (15.3%). A revision of the articles that provided information on the aetiology and the histology supports the findings of our study. In conclusion, frozen section using Feldman’s criteria had a 15.3% of false negative cases when CNS was the aetiology of the prosthetic joint infection

Aims. The outcomes of patients with unexpected positive cultures (UPCs) during revision total hip arthroplasty (THA) and total knee arthroplasty (TKA) remain unknown. The objectives of this study were to establish the prevalence and infection-free implant survival in UPCs during presumed aseptic single-stage revision THA and TKA at mid-term follow-up. Methods. This study included 297 patients undergoing presumed aseptic single-stage revision THA or TKA at a single treatment centre. All patients with at least three UPCs obtained during revision surgery were treated with minimum three months of oral antibiotics following revision surgery. The prevalence of UPCs and causative microorganisms, the recurrence of periprosthetic joint infections (PJIs), and the infection-free implant survival were established at minimum five years’ follow-up (5.1 to 12.3). Results. Of the 297 patients undergoing aseptic revisions, 37 (12.5%) had at least three UPCs obtained during surgery. The UPC cohort included 23 males (62.2%) and 14 females (37.8%), with a mean age of 71.2 years (47 to 82). Comorbidities included smoking (56.8%), hypertension (48.6%), diabetes mellitus (27.0%), and chronic renal impairment (13.5%). The causative microorganisms included Staphylococcus epidermidis (49.6%), Bacillus species (18.9%), Micrococcus species (16.2%), and Cutibacterium acnes (16.2%). None of the study patients with UPCs developed further PJIs or required further surgical intervention during follow-up. Conclusion. The prevalence of UPCs during presumed aseptic revision THA and TKA was 12.5%. The most common causative microorganisms were of low virulence, and included S. epidermidis, Bacillus species, Micrococcus species, and C. acnes. Microorganism-specific antibiotic treatment for minimum three months’ duration of UPCs in presumed aseptic revision arthroplasty was associated with excellent infection-free implant survival at mid-term follow-up. Cite this article: Bone Jt Open 2024;5(10):832–836

Aims. Currently, the most common approach for the management of a chronic PJI is a Two-Stage Replacement; because of success rates exceeding 90% when using an antibiotic impregnated cement spacer. Reliable information regarding the etiologic microorganism and its sensitivities is essential to select the antimicrobial therapy that should be used locally in the bone cement spacer during the first stage surgery as well as to select the appropriate microbiological systemic agent. Diagnostic algorithms focus to the importance of joint aspiration cultures although in the modern literature, preoperative joint aspiration has a broad range of values of sensitivity and the proportion of “dry-aspirations” is not well assessed. This low sensitivity of aspiration fluid samples in chronic-PJI is partly attributable to the fact that the majority of the microorganisms in these infections grow in biofilms attached to the implant. We have developed this biopsy technique in an effort to improve the identification rates of the causative organism. Materials and methods. A sample is harvested through a 4 mm bone trephine and the target is the bone-prosthesis gap. We have compared the results of preoperative PIB with the results of cultures from intra-operative tissue collected during the first stage surgery. In both cases a prolonged culture protocol (10 days) in enrichment media was used. On the basis of this relation, sensitivity, specificity, positive and negative predictive values and accuracy were calculated. Results. Twenty-four PIB were done on the 24 patients (10 hips and 14 knees) who subsequently underwent two-stage revision surgery because of high suspicion of PJI between January 2007 and December 2009. A retrospective analysis was performed in these 24 patients (13 women and 11 men) in the mean age of 70 years (from 63 to 88 years old). Nineteen of the cases were primary and 5 were revision arthroplasty. Nineteen patients (79%) were positive for infection from operative tissue cultures. The sensitivity was 0.79 (95% CI, 0.54–0.93); the specificity was 0.80 (95% CI, 0,30–0.99), the positive predictive value was 0.94 (95% CI, 0.67–0.99), the negative predictive value was 0.50 (95% CI, 17.5–82.5) and the accuracy was 0.79. Conclusion. PIB is a useful test to, preoperatively, isolate the infecting bacteria. The values of sensitivity, specificity and accuracy are on the average of the currently published with joint aspiration or biopsy samples cultures. Although comparative study is necessary we believe that the PIB could be useful in cases with high suspicion of PJI and negative joint aspiration cultures and in cases where no fluid is aspired from the joint, in order to preoperatively isolate the infecting bacteria

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.

Aims. This study aimed to evaluate the BioFire Joint Infection (JI) Panel in cases of hip and knee periprosthetic joint infection (PJI) where conventional microbiology is unclear, and to assess its role as a complementary intraoperative diagnostic tool. Methods. Five groups representing common microbiological scenarios in hip and knee revision arthroplasty were selected from our arthroplasty registry, prospectively maintained PJI databases, and biobank: 1) unexpected-negative cultures (UNCs), 2) unexpected-positive cultures (UPCs), 3) single-positive intraoperative cultures (SPCs), and 4) clearly septic and 5) aseptic cases. In total, 268 archived synovial fluid samples from 195 patients who underwent acute/chronic revision total hip or knee arthroplasty were included. Cases were classified according to the International Consensus Meeting 2018 criteria. JI panel evaluation of synovial fluid was performed, and the results were compared with cultures. Results. The JI panel detected microorganisms in 7/48 (14.5%) and 15/67 (22.4%) cases related to UNCs and SPCs, respectively, but not in cases of UPCs. The correlation between JI panel detection and infection classification criteria for early/late acute and chronic PJI was 46.6%, 73%, and 40%, respectively. Overall, the JI panel identified 12.6% additional microorganisms and three new species. The JI panel pathogen identification showed a sensitivity and specificity of 41.4% (95% confidence interval (CI) 33.7 to 49.5) and 91.1% (95% CI 84.7 to 94.9), respectively. In total, 19/195 (9.7%) could have been managed differently and more accurately upon JI panel evaluation. Conclusion. Despite its microbial limitation, JI panel demonstrated clinical usefulness by complementing the traditional methods based on multiple cultures, particularly in PJI with unclear microbiological results. Cite this article: Bone Joint Res 2024;13(7):353–361

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

Aims. Achievement of accurate microbiological diagnosis prior to revision is key to reducing the high rates of persistent infection after revision knee surgery. The effect of change in the microorganism between the first- and second-stage revision of total knee arthroplasty for periprosthetic joint infection (PJI) on the success of management is not clear. Methods. A two-centre retrospective cohort study was conducted to review the outcome of patients who have undergone two-stage revision for treatment of knee arthroplasty PJI, focusing specifically on isolated micro-organisms at both the first- and second-stage procedure. Patient demographics, medical, and orthopaedic history data, including postoperative outcomes and subsequent treatment, were obtained from the electronic records and medical notes. Results. The study cohort consisted of 84 patients, of whom 59.5% (n = 50) had successful eradication of their infection at a mean follow-up of 4.7 years. For the 34 patients who had recurrence of infection, 58.8% (n = 20) had a change in isolated organism, compared to 18% (n = 9) in the infection eradication group (p < 0.001). When adjusting for confound, there was no association when the growth on the second stage was the same as the first (odd ratio (OR) 2.50, 95% confidence interval (CI) 0.49 to 12.50; p = 0.269); however, when a different organism was identified at the second stage, this was independently associated with failure of treatment (OR 8.40, 95% CI 2.91 to 24.39; p < 0.001). There were no other significant differences between the two cohorts with regard to patient demographics or type of organisms isolated. Conclusion. Change in the identified microorganism between first- and second-stage revision for PJI was associated with failure of management. Identification of this change in the microorganism prior to commencement of the second stage may help target antibiotic management and could improve the success of surgery in these patients. Cite this article: Bone Jt Open 2023;4(9):720–727

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

Aims. Microbiological culture is a key element in the diagnosis of periprosthetic joint infection (PJI). However, cultures of periprosthetic tissue do not have optimal sensitivity. One of the main reasons for this is that microorganisms are not released from the tissues, either due to biofilm formation or intracellular persistence. This study aimed to optimize tissue pretreatment methods in order to improve detection of microorganisms. Methods. From December 2017 to September 2019, patients undergoing revision arthroplasty in a single centre due to PJI and aseptic failure (AF) were included, with demographic data and laboratory test results recorded prospectively. Periprosthetic tissue samples were collected intraoperatively and assigned to tissue-mechanical homogenization (T-MH), tissue-manual milling (T-MM), tissue-dithiothreitol (T-DTT) treatment, tissue-sonication (T-S), and tissue-direct culture (T-D). The yield of the microbial cultures was then analyzed. Results. A total of 46 patients were enrolled, including 28 patients in the PJI group and 18 patients in the AF group. In the PJI group, 23 cases had positive culture results via T-MH, 22 cases via T-DTT, 20 cases via T-S, 15 cases via T-MM, and 13 cases via T-D. Three cases under ongoing antibiotic treatment remained culture-negative. Five tissue samples provided the optimal yield. Any ongoing antibiotic treatment had a relevant influence on culture sensitivity, except for T-DTT. Conclusion. T-MH had the highest sensitivity. Combining T-MH with T-DTT, which requires no special equipment, may effectively improve bacterial detection in PJI. A total of five periprosthetic tissue biopsies should be sampled in revision arthroplasty for optimal detection of PJI. Cite this article: Bone Joint Res 2021;10(2):96–104

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