As the number of joint replacement procedures continues to increase worldwide, complications such as periprosthetic joint infection (PJI) are becoming more prevalent (Patel, 2023). Now recognised as the leading cause of revision surgery (Dutch Arthroplasty Register, 2024), PJI poses a major challenge to both patient outcomes and healthcare systems. It is associated with significant morbidity, including prolonged antimicrobial treatment, impaired mobility, and psychological strain. In addition, PJI imposes a substantial economic burden, involving direct costs such as repeated surgeries and extended hospitalisation (Alt et al., 2025), as well as indirect costs related to prolonged rehabilitation, caregiver dependency, and loss of productivity (Sadoghi et al., 2025; Diep et al., 2025).

Timely diagnosis of PJI is critical for effective management and improved clinical outcomes. Distinguishing infected from non-infected cases is essential, as it informs both surgical and antimicrobial treatment strategies. In patients undergoing revision surgery, rapid microbiological diagnosis enables early initiation of targeted antimicrobial therapy, thereby reducing reliance on empirical broad-spectrum antibiotics. However, conventional culture, the current gold standard, has several important limitations. Although standard culture of aerobic and facultative anaerobic microorganisms is often detected within 24–48 h, final culture results in acute PJI may still require several days and in some cases up to 1 week. In chronic PJI, extended incubation periods for anaerobic and slow-growing microorganisms may be required for up to 2 weeks (Morreel et al., 2025; Talsma et al., 2021). This can lead to potential delays in treatment decisions. Furthermore, culture may fail to detect pathogens, particularly when the causative pathogen is present in low abundance or when patients have received prior antimicrobial therapy (Esteban et al., 2025).

Molecular diagnostic techniques that detect pathogen-specific DNA or RNA have gained increasing attention as complementary tools to conventional culture (Olearo et al., 2025). By bypassing the need for microbial growth, these methods can directly identify pathogens from clinical specimens, providing a valuable alternative when culture is slow, yields false-negative results, or is affected by prior antibiotic use. Consequently, molecular diagnostics can offer substantially faster turnaround times, facilitating earlier clinical decision-making and initiation of targeted therapy.

Despite their potential, the widespread implementation of molecular diagnostic techniques has been limited by several challenges, particularly high costs and analytical complexity. In recent years, multiplex PCR assays (Gardete-Hartmann et al., 2024b; Pascual et al., 2024; Malandain et al., 2018; Sigmund et al., 2019) have been used increasingly in the field of bone and joint infections. However, the diagnostic scope of multiplex PCR is inherently constrained by the predefined targets included in the panel. As a result, clinically relevant pathogens, including coagulase-negative staphylococci and Cutibacterium acnes, may not be detected.

To overcome these limitations, molecular diagnostic methods must balance broad-range pathogen detection and diagnostic accuracy with speed and practical feasibility. Molecular Culture^® (MC) represents one such approach. This PCR-based method is based on interspace profiling (IS-pro), targeting the 16S–23S ribosomal intergenic spacer region of bacteria. This region displays species-specific length and sequence polymorphisms, enabling accurate bacterial identification. In addition, MC allows detection of polymicrobial infections and, according to the manufacturer, provides results within 4 h.

In this study, we evaluated the diagnostic performance of Molecular Culture^® using sonication fluid specimens obtained during revision surgery. Sonication fluid is a practical and suitable specimen, as sufficient sample volume is usually available, and it represents the biofilm present on the prosthetic surface. We assessed whether this approach could match or outperform conventional culture in detection of clinically relevant pathogens, with a particular focus on turnaround time and clinical applicability.

The present study demonstrates that MC provides clinically relevant pathogen detection with substantially shorter turnaround time than conventional culture. When assessed analytically using culture as a reference standard, MC demonstrated moderate positive percentage agreement (68.1 %) and high negative percentage agreement (84.6 %). However, in routine clinical practice, SF culture results are not interpreted independently. Diagnostic criteria for PJI have historically varied, yet all definitions include concordant growth of the same microorganism in two or more intraoperative samples (McNally et al., 2021; Parvizi et al., 2018; Osmon et al., 2013). In line with clinical practice, a key strength of the present study is the integration of comprehensive clinical information alongside conventional culture results to assess the clinical relevance of MC findings. By integrating prior culture results, microbiological findings from other specimen types, and relevant clinical data such as recent antibiotic exposure, MC identified 11 additional pathogens with supporting evidence of true infection. Importantly, several cases initially classified as false-positive or false-negative at the analytical level were reclassified as clinically relevant following clinical correlation (Table 2). In four of these cases, recent antibiotic exposure may have contributed to culture negativity. Consequently, incorporation of clinical data improved both PPA and, in particular, NPA (73.2 % and 95.6 %, respectively). This high clinically adjusted NPA is particularly relevant in routine practice, where distinguishing low-grade infection from aseptic failure is often challenging. As a result, patients are often treated empirically with broad-spectrum antibiotics until infection has been excluded. In this context, MC offers a means to rapidly rule out infection, allowing earlier discontinuation or avoidance of unnecessary antimicrobial therapy. Given that culture-based exclusion of infection may require up to 14 d, the short turnaround time of MC represents a clear advantage, particularly for slow-growing organisms such as Cutibacterium spp.

Several limitations should be considered. First, MC relies on equivalence sets within its identification database. In this study, results were considered concordant when the cultured microorganism belonged to the equivalence set identified by MC. However, such ambiguity may significantly influence empirical antibiotic choices. For example, differentiation between Klebsiella spp. and Enterobacter spp. is clinically relevant in our epidemiological setting, as empirical therapy would differ. In addition, MC does not provide antimicrobial susceptibility data. While conventional culture enables targeted therapy through susceptibility testing, reliance on MC alone could result in overtreatment or inappropriate therapy. This limitation is partly mitigated by adjustment of the empirical therapy based on local epidemiology but remains an important consideration for clinical implementation. False-negative MC results were observed. No consistent pattern was identified with respect to the microorganisms missed or the bacterial load, suggesting that technical factors may have contributed. Potential explanations include suboptimal DNA extraction efficiency due to sample dilution, as substantially smaller volumes are used for MC compared with culture. Furthermore, prolonged sample storage and pre-analytical factors related to sample handling may have affected overall specimen quality and DNA integrity. Finally, interpretation challenges are particularly relevant for microorganisms that may represent either true pathogens or contamination. Cutibacterium spp. are a typical example, as they are established causes of PJI but are also frequently encountered as contaminants. Although a signal threshold of ≥ 10 000 RFU was applied in this study, no uniform thresholds exist for many other organisms. Consequently, low-level contamination remains an inherent limitation of sensitive molecular techniques. Similar challenges have been described in other studies evaluating molecular methods for bone and joint infections (Olearo et al., 2025; Hong et al., 2023), where variability between techniques and the absence of standardised cut-offs complicate interpretation. Our findings reinforce the importance of clinical context and the continued need to evaluate multiple specimens and sample types to distinguish true infection from contamination.

Despite these limitations, MC demonstrated a high clinical agreement, supporting its role as a screening tool, particularly in cases where the likelihood of PJI is uncertain. Its rapid turnaround time enables earlier clinical decision-making and may facilitate timely de-escalation of empirical antimicrobial therapy. MC has been validated for use with synovial fluid and tissues, thereby enabling application across the full range of specimen types used in PJI diagnostics (Bos et al., 2023; Gardete-Hartmann et al., 2024a). It may be particularly valuable in patients who have received prior antimicrobial therapy, as certain organisms, such as streptococci, are particularly susceptible to antibiotics and therefore more likely to yield culture-negative results. Several molecular approaches have been investigated for the diagnosis of PJI, each with distinct advantages and limitations. Targeted multiplex PCR platforms offer rapid pathogen identification but are inherently dependent on the microorganisms included in the predefined panel. This limitation can be addressed by expanding target coverage or by applying broad-range approaches, such as 16S ribosomal RNA PCR or next-generation sequencing (NGS), which have demonstrated the ability to detect organisms missed by conventional culture and targeted molecular assays (Olearo et al., 2025). However, these sequencing-based approaches often require additional bioinformatic processing, specialised expertise, and longer analytical workflows, which may limit routine clinical implementation. MC may offer a balance between highly targeted assays and sequencing-based methods by combining a panbacterial approach and rapid turnaround time with a workflow that does not require complex bioinformatic analysis. Further comparative studies are required to determine the relative diagnostic performance and clinical utility of these different molecular strategies. From an implementation perspective, the relatively simple workflow of MC may facilitate integration into routine laboratory practice, making it more accessible than sequencing-based approaches. Future strategies such as sample pooling and higher-throughput testing may further improve cost-effectiveness.

In conclusion, MC provides rapid and clinically relevant information for the diagnosis of PJI, particularly in cases where infection needs to be excluded or where prior antibiotic exposure limits the sensitivity of conventional culture. Its clinical value lies in its use as a complementary tool, requiring careful interpretation within the clinical context. In contrast to approaches that position molecular diagnostics as a rescue test, our findings support early integration of MC in the diagnostic pathway. By enabling prompt exclusion of infection, early MC testing may reduce unnecessary empirical (intravenous) antibiotic use, shorten hospitalisation, and decrease healthcare costs. From an antimicrobial stewardship perspective, this strategy has the potential to optimise both diagnostic efficiency and downstream clinical management.