Aims. The microbiological detection of microorganisms plays a crucial role in the diagnosis as well as in the targeted systemic and local antibiotic therapy of periprosthetic infections (PJI). Despite extensive efforts to improve the sensitivity of current culture methods, the rate of culture-negative infections is approximately 10–20% of all PJI. This study investigates an preanalytical algorithm (culture collection and direct processing in the OR) to potentially increasing culture yield in patients with PJI. Methods. Patients undergoing staged revision arthroplasty for PJI in our hospital between October 2021 and 2022 were included in this prospective pilot study. Intraoperatively twenty tissue samples were collected and distributed among 4 groups. Tissue samples were prepared according to standard without medium and in thioglycolate medium at 3 different temperatures (room temperature, 4°C, 37° for 24h before transport to microbiology) directly in the OR. The removed implants were sonicated. Cultures were investigated on days 1, 3, 7, 12, 14 for possible growth. All grown organism, the number of positive samples and the time to positivity were recorded and compared. Results. 71 patients were included (age, gender). Compared to the standard procedure the thioglycolate broth at 37°C was significantly more often culture-negative (p=0.031). No significant differences in the frequency of culture-negative samples were detected in the other groups. 8.4% (6/71) patients were culture negative in the standard culture but positive in the thioglycolate samples. In contrast, 7% (5/71) were culture negative in the thioglycolate samples but had bacterial detection in the standard approach. In 4.7% (3/63) of the patients, only the sonication showed growth, whereas 25.4% (16/63) had no growth in sonication fluid but in one of the cultures. For S. caprae, there was a significantly different distribution (p=0.026) with more frequent detection in the group with thioglycolate at 37°C. The standard procedure (p=0.005) and sonication (p=0.023) showed a shorter time to positivity of the culture compared to the thioglycolate approach at 4°C. Conclusions. No general differences could be shown between the standard preparation and the thioglycolate preparation; in particular, storage at different temperatures does not seem to result in any difference. For individual cases (8% in this study), bacterial growth was detected in the thioglycolate group that would have been culture-negative otherwise. There might be organism dependent differences in growth in different media

Aims. To identify the most common infecting organisms associated with deep infection and infected non-union of the tibia, as well as the rate of ‘culture-negative’ infections, at a tertiary referral trauma centre dealing with military and civilian trauma. Method. Between 2008 and 2010 all patients with a confirmed clinical diagnosis of implant-related infection or infected non-union of the tibia were identified retrospectively from a database and their records analysed. After a period of at least 10–14 days without antibiotics, all patients underwent surgical debridement in which ‘clean’ samples were went for microbiological analysis. Skeletal stablity was achieved with a circular frame and intravenous antibiotics were started pending culture and sensitivity results. Results. There were 31 patients; 28 male, 3 female. Mean age 41yrs. 21 patients (68%) had sustained open fractures. In 11 patients, tissue cultures were negative. In the remainder, 8 patients cultured one organism, 6 two organisms, 2 three organisms, and 2 four organisms. Only those with open fractures grew more than 2 organisms. A total of 15 different infecting organisms (all of which were bacteria) were identified. The most common organisms isolated were Staphylococcus aureus, coagulase-negative Staphylococcus, Bacillus and Enterobacter cloacae. There were 3 cases of MRSA infection, all in open fractures. The most frequently used antibiotics for definitive therapy were ciprofloxacin, rifampicin and vancomycin. Conclusions. The majority of patients had sustained open fractures. Patient with open fractures were more likely to have polymicrobial infections. There was a high rate of culture-negative infections, particularly in the closed fracture group. Meticulous sampling and processing of microbiology samples is essential to minimise the number of culture-negative infections. Laboratory techniques such as polymerase chain reaction may help improve culture yields

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

Aim. Spinal implant-associated infections (SIAI) require combined surgical and antimicrobial treatment and prolonged hospital stay. We evaluated the clinical, laboratory, microbiological and radiological characteristics and treatment approaches in patients with SIAI. Method. Consecutive adult patients with SIAI treated between 2015 and 2017 were prosepctively included. SIAI was defined by: (i) significant microbial growth from intraoperative tissue or sonication fluid, (ii) intraoperative purulence, secondary wound dehiscence or implant on view, (iii) radiographic evidence of infection and fever (>38°C) without other recognized cause, increasing back pain or neurologic impairment, (iv) peri-implant tissue inflammation in histopathology. Results. A total of 60 patients were included, median age was 66 years (range, 28–91 years), 29 (48%) were males. The most common reason for spinal stabilization was spinal canal stenosis in 20 patients (33%) followed by vertebral degenerative disease in 14 (23%). 31 patients (52%) had one or more previous spine surgeries (range, 1–4 interventions). The anatomic site of spinal instrumentation was lumbar/sacral in 26 (43%), cervical in 23 patients (38%), thoracic in 11 (18%). The median number of fused segments was 5 (range, 1–14). Clinical manifestations included wound healing disturbance in 41 patients (68%), increasing back pain in 15 (25%), neurologic impairment in 12 patients (20%) and fever in 14 (23%). Serum CRP was abnormal (>10mg/l) in 46/59 patients (78%). Most (n=54) infections were postsurgical, 5 were hematogenous and 1 was contiguous. Imaging showed epidural, intraspinal or paravertebral abscess in 21/42 patients (50%), implant failure in 9 (21%) and implant loosening in 3 cases (7%). Monomicrobial infection was observed in 41 (68%), polymicrobial in 16 (27%) patients and culture-negative infection in 3 episodes (5%). Predominant causative pathogens were S. aureus (n=19), coagulase-negative staphylococci (n=18) and gram-negative rods (n=16). Surgery was performed in all patients including debridement and implant retention in 39 patients (65%), partial implant exchange in 10 (17%) and complete exchange in 11 (18%). Antimicrobial treatment included biofilm-active substances in 52 patients (87%). The median duration of antimicrobial therapy was 11,7 weeks (range, 6–12 weeks). 14 patients (23%) recieved suppressive therapy for a median duration of 9 months (range 3–12 months). Conclusions. Most SIAI were seen in lumbar/sacral segments and wound healing disturbance and increasing back pain were the most common manifestations. In 95% the causative pathogen was isolated, predominantly staphylococci. In half of the episodes, abscesses were present. All patients underwent surgery and biofilm-active antibiotics were administered in 87%

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.