# Synovial Biomarkers C-Reactive Protein and Calprotectin for Diagnosing Chronic Periprosthetic Joint Infection: A Prospective Multicenter Evaluation

Louise Ruffier d'Epenoux, Manon Robert, Raphaël Lecomte, Christophe Nich, Pascale Bémer, Stéphane Corvec

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Louise Ruffier d'Epenoux, Manon Robert, Raphaël Lecomte, Christophe Nich, Pascale Bémer, et al. Synovial Biomarkers C-Reactive Protein and Calprotectin for Diagnosing Chronic Periprosthetic Joint Infection: A Prospective Multicenter Evaluation. The Journal of Arthroplasty, 2025, S0883-5403 (25), 10.1016/j.arth.2025.06.078. inserm-05163509

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# Synovial Biomarkers C-Reactive Protein and Calprotectin for Diagnosing Chronic Periprosthetic Joint Infection: A Prospective Multicenter Evaluation

Louise Ruffier d'Epenoux, PharmD<sup>a, b, c</sup>, Manon Robert, PharmD<sup>d</sup>, Raphaël Lecomte, MD<sup>c, e, f</sup>, Christophe Nich, MD, PhD<sup>c, g, h</sup>, Pascale Bémer, MD<sup>a</sup>, Stéphane Corvec, PharmD, PhD<sup>a, b, c, i, *</sup>, on behalf the CRIOGO/CALPROS Study group<sup>1</sup>

## ARTICLE INFO

**Article history:**

Received 15 March 2025

Received in revised form

26 June 2025

Accepted 27 June 2025

Available online xxx

**Keywords:**

biomarker

synovial fluid

C-reactive protein

calprotectin

periprosthetic joint infections

## ABSTRACT

**Background:** The diagnosis of periprosthetic joint infection (PJI) remains a challenge in clinical practice, and synovial fluid (SF) analysis is a valuable diagnostic tool. Recently, two synovial biomarkers (leukocyte esterase strip test and alpha-defensin [AD]) have been incorporated within the MSIS (Musculoskeletal Infection Society) algorithm for PJI diagnosis. Although AD is promising, with high sensitivity and specificity, it remains expensive. Calprotectin, another protein released upon activation of articular neutrophils, offers a low-cost alternative. Calprotectin and CRP (serum C-reactive protein) joint determination is now feasible in routine laboratory practice. This study aimed to evaluate the diagnostic performance of two synovial biomarkers, calprotectin and CRP, for PJI diagnosis.

**Methods:** From April 2022 to June 2024, all adult patients undergoing preoperative or intraoperative aspiration of hip or knee prosthetic joints were included in this prospective, multicenter, noninterventional study. Patients who had prosthetic implantation less than three months prior or those who had systemic inflammatory diseases were excluded. For each SF sample, calprotectin and CRP synovial measurements were performed. Patients were classified as infected (PJI group) or noninfected (non-PJI group) based on decisions made during multidisciplinary meetings. During the study period, 157 patients were enrolled. There were 59 patients excluded due to insufficient synovial fluid volume or failure to meet inclusion criteria. There were 52 patients classified into the PJI group and 46 into the non-PJI group.

**Results:** The sensitivity, specificity, positive likelihood ratio and negative likelihood ratio of synovial calprotectin were 94.23%, 78.26%, 4.24, and 0.07, respectively, while those of synovial CRP were 80.77%, 86.96%, 6.19, and 0.22, respectively.

**Funding:** This research project was carried out as part of our routine work and received no external funding.

One or more of the authors of this paper have disclosed potential or pertinent conflicts of interest, which may include receipt of payment, either direct or indirect, institutional support, or association with an entity in the biomedical field which may be perceived to have potential conflict of interest with this work. For full disclosure statements refer to https://doi.org/10.1016/j.arth.2025.06.078.

* Address correspondence to: Stéphane Corvec, PharmD, PhD, Institut de Biologie des Hopitaux de Nantes, Service de Bactériologie et des Contrôles Microbiologiques, CHU de Nantes, 9 quai Moncousu, Nantes 44093, France.

¹ CRIOGO/CALPROS Study group: Rachel Chenouard, Hélène Pailhories, Didier Tandé, Claudie Lamoureux, Anaïs Greves, Gaëlle Novert, Sévérine Ansart, Luc Quaesaet, Pierre Gazeau, Thomas Williams, Antoine Desseaux, Barbara Plantard, Raphaël Lecomte, Christophe Nich, Vincent Crenn, Pascale Bémer, Stéphane Corvec, Louise Ruffier d'Epenoux, Charles Hervochon, Axelle Paquin, Barbara Plantard, Antoine Asquier Khati, David Butoille, Hélène Caillon, Thomas Dejoie, Chloé Plouzeau-Jaye, Sophie Reissier, Harold Common, Marion Baldeyrou, Solene Patrat-Delon, Marie-Frédérique Lartigue, Laura Chaufour, Antoine Tesson, Louis Romée Le Nail, Geoffroy Dubois de Montmarin, Adrien Lemaignen, Marion Lacasse, Vianney Tuloup, Eve-Marie Takoudju.

<footnote><sup>a</sup> Service de Bactériologie et des Contrôles Microbiologiques, CHU Nantes, Nantes, France</footnote>

<footnote><sup>b</sup> INSERM, Immunology and New Concepts in ImmunoTherapy, INCIT, UMR 1302, Nantes Université, Nantes, France</footnote>

<footnote><sup>c</sup> Membre du CRIOGO (Centre de Référence des Infections Ostéo-articulaires du Grand Ouest), Nantes, France</footnote>

<footnote><sup>d</sup> Service de Biochimie, CHU Nantes, Nantes, France</footnote>

<footnote><sup>e</sup> Service des Maladies Infectieuses, Hôtel-Dieu, Centre Hospitalier Universitaire, Nantes, France</footnote>

<footnote><sup>f</sup> Centre d'Investigation Clinique Unité d'Investigation Clinique, Centre Hospitalier Universitaire, Nantes, France</footnote>

<footnote><sup>g</sup> Nantes Université, CHU Nantes, Clinique Chirurgicale Orthopédique et Traumatologique, Nantes, France</footnote>

<footnote><sup>h</sup> Nantes Université, INSERM, UMRS, 1229, Regeneration Medicine and Skeleton (RMeS), ONIRIS, Nantes, France</footnote>

<footnote><sup>i</sup> ESGIAI (ESCMID Study Group for Implant-Associated Infections) member from Service de Bactériologie, CHU Nantes, Nantes, France</footnote>
**Conclusions:** The present study demonstrates that synovial calprotectin and synovial CRP assays have excellent accuracy for the diagnosis of PJL. These findings suggest that these two synovial biomarkers should be incorporated into a new decision-making algorithm for PJL diagnosis.

© 2025 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Joint arthroplasty is a common procedure that tremendously improves patients' quality of life. However, up to 10% of these devices may encounter complications over the patient's lifetime [1]. Among these, periprosthetic joint infections (PJIs) are one of the most severe and prevalent, occurring in less than 1 to 2% of primary arthroplasties [2–5]. The PJIs pose a major burden both to patients and health care systems due to the need for complex management and prolonged hospital stays [6,7]. They can result in poor functional outcomes and, in some cases, lead to permanent disability [2]. *Staphylococcus aureus* and coagulase-negative staphylococci are the most commonly identified pathogens, responsible for 30 to 43% of cases, respectively, followed by mixed flora (10 to 11%), streptococci (9 to 10%), Gram-negative bacilli (3 to 6%), enterococci (3 to 7%), and anaerobes (2 to 4%) [2,8].

The identification, eradication, and treatment of PJIs are particularly challenging due to their complex pathogenesis, driven by microbial growth within biofilms and microcolonies [9].

Important advancements have been made in the PJL's bacteriological management, including the bacterial extraction via bead mill processing or sonication, the inoculation of four samples on three culture media, and the use of blood culture bottles. Despite these improvements, 5 to 15% of suspected PJIs remain sterile in bacteriological cultures [10].

In recent years, several organizations, including the Musculoskeletal Infection Society (MSIS) [11] and the European Bone and Joint Infection Society (EBJIS) [12] have published standardized definitions for the diagnosis of PJL. Among the minor criteria, synovial fluid biomarkers such as alpha-defensin and leukocyte esterase (LE) have gained prominence. Alpha-defensin, in particular, has demonstrated consistently high specificity [13–15], but it may yield false-positive results in noninfectious inflammatory conditions like metallosis, gout, or inflammatory diseases [16–18]. Consequently, a negative test should not be solely relied upon to exclude PJL, given its low sensitivity [13,19]. In a recent metaanalysis, LE was found to have diagnostic accuracy similar to that of positive culture bacteriology or synovial white cell counts. Moreover, in the case of a hemorrhagic sample, this colorimetric parameter becomes uninterpretable, further limiting its clinical relevance [20].

An emerging synovial fluid biomarker is calprotectin. This cytoplasmic protein, predominantly expressed in neutrophils, is released into the extracellular environment following neutrophil activation and exhibits antimicrobial properties [21]. Fecal calprotectin has long been used in the diagnosis and the monitoring of inflammatory bowel disease (IBD) [22], and more recently, it has gained attention in the diagnosis of PJL. This test is cost-effective and user-friendly compared to other biomarkers. Several studies have reported high sensitivity and specificity for synovial calprotectin in detecting PJIs [23,24].

The objective of this prospective multicenter study was to assess the diagnostic performance of joint calprotectin and C-reactive protein (CRP) for detecting PJIs. Our research was conducted through the CRIOGO (Centre de Référence des Infections Ostéo-articulaires du Grand Ouest, the western part of France) network of seven reference centers specialized in the multidisciplinary diagnosis and treatment of PJIs [25,26]. This study is a multicenter prospective investigation focusing on the use of these synovial biomarkers for PJL diagnosis and, to our

knowledge, for the first time, proposes a diagnostic algorithm integrating these biomarkers.

## Materials and Methods

### Ethics Approval

This study received ethical approval from our hospital's Institutional Review Board and was consistent with the Declaration of Helsinki. Each patient gave informed consent for anonymized data use for research purposes before the interventional procedure. Our database, including laboratory and surgical data, was anonymized in accordance with the French Data Protection Regulation. This study was recorded at Nantes University Hospital by the local Data Privacy Officer under the reference 22-3-131.

### Patients and Study Design

All adult patients (over 18 years of age) undergoing pre- or intraoperative aspiration of hip or knee prosthesis were included. Patients who had systemic inflammatory diseases (such as rheumatoid arthritis, ankylosing spondylitis, or chronic inflammatory bowel disease) and those who had undergone prosthesis implantation within the past three months were excluded. Following surgery, a multidisciplinary decision meeting (CRIOGO group) was held, involving systematically a surgeon, an infectious disease specialist, and a microbiologist, to classify patients as either infected (PJL group) or noninfected (non-PJL group). Synovial biomarker analysis and clinical evaluation were conducted independently to prevent any potential bias. Consequently, the synovial biomarker values were not disclosed during the multidisciplinary decision-making meeting.

Synovial fluid (SF) samples were collected either in dry tubes with no chemical preservative (for SF cytology) or in heparinized tubes (for synovial biomarker assays). The SF cytology and sample preparation were conducted at each center, while synovial biomarker assays were centralized and performed exclusively at Nantes University Hospital. Cell counting of SF was done manually, using 10 cell count slides with grids (Biosigma, Cantarana di Cona, Italy). The cells were quantified under x40 magnification. The results were reported as the number of cells per microliter. In one center, the leukocyte count was performed using the UF 4000 automated system (Sysmex Co., Kobe, Japan), following a prior 1:10 dilution in saline solution. All samples were centrifuged at 3,500 rpm for five minutes, after which the supernatant was collected for the determination of leukocyte esterase, CRP, and calprotectin measurements. Lyophilized powder of hyaluronidase (Sigma-Aldrich, Saint Louis, USA) was utilized to liquefy the samples prior to the calprotectin assay.

### Patient Cohort

Between April 1, 2022 and August 30, 2024, 157 patients who met the inclusion criteria were screened for enrollment in this prospective multicenter study. There were 59 patients excluded for reasons including insufficient SF (n = 33), technical issues (n = 9), and failure to meet the inclusion criteria (n = 17). Ultimately, 98 patients were enrolled (Figure 1). Of the 98 patients included, 52 were classified as
<figure><img src="image_5.png" /><figcaption>**Figure 1.** Study flow chart of patients included. PJI, periprosthetic joint infection.</figcaption></figure>

infected and 46 as noninfected. The initial surgical management of PJI among the 98 patients was diagnostic aspiration in 27 (27.6% [11 patients in the PJI group and 16 patients in the non-PJI group]); debridement with implant retention in 12 (12.2%; exclusively in the PJI group); one-stage implant retention in 49 (50% [22 patients in the PJI group and 27 patients in the non-PJI group]); two-stage implant exchange in six (6.1% [four patients in the PJI group and two patients in the non-PJI group]); and other procedures in 4 (4.1% [three patients in the PJI group and one patient in the non-PJI group]) (Table 1). All the patients included in this study had chronic infection. Generally, this infection is considered a contraindication to "debridement with implant retention." The 12 patients who underwent this procedure were hemodynamically too unstable to undergo a one-stage or two-stage implant exchange.

The non-PJI patient group consisted of 22 men and 24 women, who had a mean age of 69 years (range, 33 to 92) and a mean body mass index of 27.7. The majority of cases were total hip arthroplasties (THAs) (56.5%) and total knee arthroplasties (TKAs) (43.5%). In nearly half of the patients, the primary clinical presentation was radiological loosening. All patients in this group had negative culture results (Table 1).

In the PJI group, there were 40 men and 12 women, who had a mean age of 69 years (range, 26 to 95) and a mean body mass index of 27.9. A total of 31 patients had failed THAs, and 21 had failed TKAs. Positive culture results were found in 41 patients, with six cases exhibiting polymicrobial infections. The isolated pathogens included coagulase-negative staphylococci (n = 21), *S. aureus* (n = 9), *Streptococcus* species (n = 6), Gram-negative microorganisms (n = 7), anaerobic bacteria (n = 6), and *Mycobacterium tuberculosis* (n = 1) (Table 1).

### Synovial fluid leukocyte esterase (sLE)

The LE strip test used in this study was the Multistix 8SG urine strip test (Siemens, Tarrytown, New York, USA). We defined a 2+ result to be positive for PJI [24].

### Synovial fluid C-reactive protein (sCRP)

For the index tests, sCRP was measured by immunoturbidimetry on a Cobas Pro c503 analyzer (Roche Diagnostic, Meylan,

France). The determination of sCRP was based on an immunoturbidimetric assay. The sCRP test was considered suggestive for PJI when > 8.8 mg/L, as previously published by Gallo et al. [27].

### Synovial Fluid Calprotectin

Calprotectin determination was performed using the Bühlmann fCAL turbo reagents on the Optilite platform (The Binding Site—part of Thermo Fisher Scientific, Birmingham, United Kingdom). This assay is based on a particle-enhanced turbidimetric immunoassay (PETIA), which uses antibodies covalently coupled to latex particles to quantify calprotectin in fecal samples with high precision and reproducibility in a fully automated process. The calprotectin test was considered suggestive for PJI when >50 mg/L, as previously published by Wouthuyzen-Bakker et al. [23].

### Culture

The SF and tissue specimens were processed by the laboratory within two hours of the collection according to the standardized laboratory protocols for aerobic and anaerobic cultivation in PJI. Bacterial extraction via bead mill processing was used in all centers [26]. Bacterial extraction using stainless steel beads on the Retsch MM400 bead mill (Retsch GmbH, Haan, Germany) for 2.5 min at 30 Hz was performed in all centers.

### Data Analyses

Statistical analyses were performed using EXCEL (Microsoft Corporation, Redmond, Washington, USA) and XLSTAT (Addinsoft, Paris, France). A higher area under the curve (AUC) indicated better discriminatory performance: 0.5 to 0.6 (no accuracy), 0.6 to 0.7 (poor accuracy), 0.7 to 0.8 (moderate accuracy), and 0.9 to 1.0 (excellent accuracy). A P-value of < 0.05 was used to define statistical significance.

## Results

The median sCRP levels were significantly higher in patients who had PJI, at 27.7 mg/L (interquartile range [IQR] 16.9 to 58.7),
<caption>**Table 1**</caption>

Demographic, Clinical, and Biological Characteristics of the Patients Included in the Trial<sup>a</sup>

<table><thead><tr><th>Patients' Characteristics</th><th>Overall (N = 98)</th><th>PJI Group (N = 52)</th><th>Non-PJI Group (N = 46)</th></tr></thead><tbody><tr><td><strong>Demographic Characteristic</strong></td><td></td><td></td><td></td></tr><tr><td>Age—year</td><td>69 ± 14</td><td>69 ± 16</td><td>69 ± 13</td></tr><tr><td>Men—no./total no. (%)</td><td>62/98 (63.3)</td><td>40/52 (76.9)</td><td>22/46 (47.8)</td></tr><tr><td><strong>Clinical Characteristic</strong></td><td></td><td></td><td></td></tr><tr><td>History of prosthetic joint infection—no./total no. (%)<sup>b</sup></td><td>31/98 (31.6)</td><td>24/52 (46.2)</td><td>7/46 (15.2)</td></tr><tr><td>Baseline surgical procedure—no./total no.(%)</td><td></td><td></td><td></td></tr><tr><td>Diagnostic aspiration</td><td>27/98 (27.6)</td><td>11/52 (21.2)</td><td>16/46 (34.8)</td></tr><tr><td>Debridement with implant retention</td><td>12/98 (12.2)</td><td>12/52 (23.1)</td><td>0/46 (0)</td></tr><tr><td>One-stage prosthetic joint implant exchange</td><td>49/98 (50)</td><td>22/52 (42.3)</td><td>27/46 (58.7)</td></tr><tr><td>Two-stage prosthetic joint implant exchange</td><td>6/98 (6.1)</td><td>4/52 (7.7)</td><td>2/46 (4.3)</td></tr><tr><td>Other<sup>c</sup></td><td>4/98 (4.1)</td><td>3/52 (5.8)</td><td>1/46 (2.2)</td></tr><tr><td>Affected joint—no./total no. (%)</td><td></td><td></td><td></td></tr><tr><td>Hip</td><td>57/98 (58.2)</td><td>31/52 (59.6)</td><td>26/46 (56.5)</td></tr><tr><td>Knee</td><td>41/98 (41.8)</td><td>21/52 (40.4)</td><td>20/46 (43.5)</td></tr><tr><td>Body-mass index<sup>d</sup></td><td>27.8 ± 5.0</td><td>27.9 ± 4.6</td><td>27.7 ± 5.4</td></tr><tr><td>Coexisting medical condition—no./total no. (%)</td><td></td><td></td><td></td></tr><tr><td>Obesity</td><td>25/98 (25.5)</td><td>10/52 (19.2)</td><td>15/46 (32.6)</td></tr><tr><td>Clinical presentation—no./total no. (%)</td><td></td><td></td><td></td></tr><tr><td>Fever</td><td>13/98 (13.3)</td><td>13/52 (25.0)</td><td>0/46 (0)</td></tr><tr><td>Fistula</td><td>10/98 (10.2)</td><td>10/52 (19.2)</td><td>0/46 (0)</td></tr><tr><td>Wound dehiscence</td><td>3/98 (3.1)</td><td>3/52 (5.8)</td><td>0/46 (0)</td></tr><tr><td>Radiological loosening</td><td>28/98 (28.6)</td><td>7/52 (13.5)</td><td>21/46 (45.7)</td></tr><tr><td>Median time between symptom onset and surgical procedure (IQR)—month<sup>e</sup></td><td>72 (22 to 168)</td><td>60 (19.5 to 159)</td><td>96 (23 to 168)</td></tr><tr><td><strong>Biological characteristics</strong></td><td></td><td></td><td></td></tr><tr><td>Serum CRP level at diagnosis of infection—mg/L<sup>f</sup></td><td>64.7 ± 93.2</td><td>103.5 ± 106.7</td><td>17.5 ± 35.6</td></tr><tr><td>Synovial White Blood Cell Count—/mm<sup>3g</sup></td><td>23,017.1 ± 65,851.0</td><td>40,821.9 ± 83,636.1</td><td>1596.9 ± 4839.5</td></tr><tr><td>Positive blood culture—no./total no. (%)<sup>h</sup></td><td>8/98 (8.2)</td><td>7/52 (13.5)</td><td>1/46 (2.2)</td></tr><tr><td>Positive bacterial culture—no./total no.(%)</td><td>41/98 (41.8)</td><td>41/52 (78.8)</td><td>0/46 (0)</td></tr><tr><td>Mono-microorganism—no./total no. (%)<sup>i</sup></td><td>35/98 (35.7)</td><td>35/52 (67.3)</td><td>0/46 (0)</td></tr><tr><td>Pathogens identified—no./total no.(%)</td><td></td><td></td><td></td></tr><tr><td>Staphylococcus aureus</td><td>9/50 (18.0)</td><td>9/50 (18.0)</td><td>0/50 (0)</td></tr><tr><td>Coagulase-negative staphylococcus</td><td>21/50 (42.0)</td><td>21/50 (42.0)</td><td>0/50 (0)</td></tr><tr><td>Streptococcus species</td><td>6/50 (12)</td><td>6/50 (12)</td><td>0/50 (0)</td></tr><tr><td>Gram-negative organisms</td><td>7/50 (14)</td><td>7/50 (14)</td><td>0/50 (0)</td></tr><tr><td>Anaerobic bacteria</td><td>6/50 (12)</td><td>6/50 (12)</td><td>0/50 (0)</td></tr><tr><td>Mycobacterium tuberculosis</td><td>1/50 (2)</td><td>1/50 (2)</td><td>0/50 (0)</td></tr></tbody></table>

PJI, Periprosthetic Joint Infection.

a Plus-minus values are means ± SD. IQR denotes interquartile range.

b History of prosthetic joint infection was defined as having at least one previous episode.

c Other procedures included irrigation and debridement without exchange of molecular mobile components, irrigation with fistulization, arthrodesis, and unipolar exchange of the femoral head.

d Body-mass index (BMI) is the weight in kilograms divided by the square of the height in meters. Obesity was defined by BMI greater than 30.

e Data on time between symptom onset and surgical procedure were available for 47 patients in the non-PJI group.

f Data on CRP level at diagnosis of infection are available for 47 patients in the non-PJI group and 52 patients in the PJI group.

g Data on Synovial White Blood Cell Count were available for 51 patients in the PJI group and 45 patients in the non-PJI group.

h Data on positive blood culture are available for 47 patients in the non-PJI group and 52 patients in the PJI group.

i A total of 6 patients in the PJI group had a polymicrobial infection.

compared to 0.7 mg/L (IQR 0.2 to 2.0) in the non-PJI group (Figure 2). Similarly, the median synovial calprotectin levels were markedly elevated in the PJI group, with a median value of 400 mg/L (IQR 400 to 400), versus 8.3 mg/L (IQR 0 to 33.9) in the non-PJI group. Additionally, the median sLE in patients who had PJI was 3+ (IQR 2+ to 3+), compared to 1+ (IQR 0 to 2+) in the non-PJI group.

Synovial fluid calprotectin and sCRP had excellent diagnostic strength with AUCs of 0.92 and 0.94, respectively (Figure 3). Sensitivity and specificity were 94.2 and 78.3% for calprotectin (cut-off value 50 mg/L) and 80.8 and 87.0% for sCRP (Table 2). When combining synovial calprotectin and sCRP to diagnose PJI, the AUC was higher than that obtained when using sCRP alone (AUC = 0.96 versus 0.94) or synovial calprotectin alone (AUC = 0.96 versus 0.92).

Discussion

The microbiological diagnosis of prosthetic osteoarticular infections remains a major clinical challenge, requiring extensive

expertise. Despite considerable advancements in bacteriological sample processing [26,28,29], cultures yield negative results in 5 to 15% of cases [3,4], even when clinical findings strongly suggest infection. In such cases, incorporating molecular techniques or evaluating synovial biomarkers may provide critical diagnostic insights. In 2018, Parvizi et al. [11] included the synovial biomarkers alpha-defensin and leukocyte esterase in their diagnostic criteria guidelines for periprosthetic joint infection. Alpha-defensin can be detected either through an enzyme-linked immunosorbent assay (ELISA) or using a commercially available test kit. Although this biomarker demonstrates high sensitivity and specificity [30,31], false positives have been reported, particularly in cases of metallosis [17]. Furthermore, the high cost of the assay limits its widespread application in routine clinical practice. Measurement of synovial leukocyte esterase is straightforward, cost-effective, and widely available in our laboratories. However, as it relies on a colorimetric assay, there is substantial interoperator variability. Additionally, this test is not applicable in cases of hemorrhagic SF. Consequently, in this multicenter prospective study, we aimed to evaluate alternative synovial biomarkers
<figure><img src="image_6.png" /><figcaption>**Figure 2.** Synovial Levels of A) Calprotectin, B) C-Reactive Protein, and C) Leukocyte Esterase in Patients in the PJI and Non-PJI groups, Data on synovial calprotectin and C-reactive protein were available for all patients. Leukocyte esterase data were available for 49 patients in the PJI group and 40 patients in the non-PJI group. PJI, periprosthetic joint infection.</figcaption></figure>

(synovial calprotectin and synovial CRP) for the diagnosis of chronic osteoarticular infections in prosthetic joints.

Our data demonstrate that synovial calprotectin and sCRP are sensitive and specific biomarkers that help in the diagnosis of PJIs. With an NLR of 0.07, synovial calprotectin is particularly useful for excluding PJI in patients who have chronic pain. However, calprotectin may still yield false-negative results in a minority of patients who have low-grade infections, as revealed in three out of 52 patients in this study. In one of these cases, calprotectin was measured during the second phase of a two-stage exchange procedure (at the reimplantation step) following a median period of 12 weeks without antibiotics. There were three intraoperative tissue cultures positive for *Cutibacterium acnes*, suggesting persistent infection or reinfection. Serum CRP, synovial CRP, and synovial white blood cell count were all false negatives. This finding has also been demonstrated by Wouthuyzen-Bakker et al. [23] The

second patient presented with a relapse three years after the initial infection, caused by *C. acnes* (single-locus sequence typing (SLST) [32] showed identical SLST-types for both strains). In these two cases, probably due to the low bacterial inoculum [23] and the potential integration within osteoblasts [33], the synovial biomarker assays seem to lack sufficient sensitivity to detect the infection.

Also, the third case is a complex one. The disease history began in January 2022 with right hip pain. Preoperative aspiration revealed Gram-positive cocci; however, no pathogen was cultured. 16S rRNA PCR (primer set 27F/16S1RRB) [34] identified *Gemmibacter formicilis* and *Subdoligranulum variabile* (anaerobic non-cultivable pathogens). In February 2024, the patient was seen again in consultation due to a febrile episode and severe hip pain associated with functional impairment. A lavage and microbiological samples were taken, but no pathogen was identified either

<figure><img src="image_7.png" /><figcaption>**Figure 3.** AUC of A) Calprotectin, B) sC-Reactive Protein, C) Leukocyte Esterase synovial tests, and D) Combined score synovial CRP and calprotectin, Data on synovial calprotectin, CRP and combined score were available for all patients. Leukocyte esterase data were available for 49 patients in the PJI group and 40 patients in the non-PJI group. PJI, periprosthetic joint infection; sCRP, synovial CRP; AUC, area under the curve.</figcaption></figure>
<table><caption>**Table 2**
Diagnostic Accuracy of Synovial Calprotectin, C-Reactive Protein, and Leukocyte Esterase.</caption><thead><tr><th>Test</th><th>Cutoff Value</th><th>Sensitivity %</th><th>Specificity %</th><th>PLR</th><th>NLR</th><th>AUC</th></tr></thead><tbody><tr><td>Calprotectin</td><td>50 mg/L</td><td>94.23% (95% CI 84.3 to 98.4)</td><td>78.26% (95% CI 64.4 to 87.7)</td><td>4.34</td><td>0.07</td><td>0.92 (95% CI 0.85 to 0.98)</td></tr><tr><td>Calprotectin</td><td>80 mg/L</td><td>94.23% (95% CI 84.3 to 98.4)</td><td>84.78% (95% CI 71.8 to 92.4)</td><td>6.19</td><td>0.07</td><td></td></tr><tr><td>CRP</td><td>8.8 mg/L</td><td>80.77% (95% CI 68.1 to 89.2)</td><td>86.96% (95% CI 74.3 to 93.9)</td><td>6.19</td><td>0.22</td><td>0.94 (95% CI 0.89 to 0.98)</td></tr><tr><td>Leukocyte esterase</td><td>2+</td><td>67.4% (95% CI 53.4 to 78.8)</td><td>92.50% (95% CI 80.1 to 97.4)</td><td>8.98</td><td>0.35</td><td>0.83 (95% CI 0.75 to 0.92)</td></tr></tbody></table>

Data on synovial calprotectin and C-reactive protein were available for all patients. Leukocyte esterase data were available for 49 patients in the PJI group and 40 patients in the non-PJI group.

PJI, prosthetic joint infection; PLR, positive likelihood ratio; NLR, negative likelihood ratio; AUC, area under the ROC curve; CI, confidence interval.

by culture or 16S rRNA PCR. Nevertheless, the patient was diagnosed by the multidisciplinary team as having a PJI.

The primary advantages of using synovial calprotectin and sCRP as biomarkers in clinical practice, compared to other available options, are (i) their availability, as they are already employed in routine care for other purposes in most hospitals; (ii) the ability to provide a quantitative measurement; and (iii) their low cost (10.60 euros [approximately 10.95 USD] per sample for synovial

calprotectin and 0.38 euros [approximately 0.39 USD] per sample for sCRP).

There were several potential limitations in the present study. The determination of infection was based on a multidisciplinary decision-making meeting (CRIOGO group), which included a surgeon, an infectious disease specialist, and a microbiologist. Additionally, we did not apply the MSIS 2018 score, as we did not have access to all the minor criteria (alpha-defensin, ESR, and

<figure><img src="image_8.png" /><figcaption>**Figure 4.** Proposal of an algorithm for routine Periprosthetic joint infection diagnosis. PJI, periprosthetic joint infection; sCalpro, synovial calprotectin; sCRP, synovial CRP; sLE, synovial leukocyte esterase; CRP, serum C-reactive protein; sWB, synovial white blood cell count; MSIS, Musculoskeletal Infection Society.</figcaption></figure>
histology). Another limitation is the relatively modest sample size, despite it being larger than most previously published cohorts using ELISA for synovial calprotectin measurement [35]. Future studies with larger populations or registry-based designs are needed to validate and generalize our findings. An additional limitation of our study is the relatively small size of the subgroups. The number of patients should be increased in future studies to analyze the impact of prior antibiotic use, immunosuppressants, as well as metallosis on the diagnostic performance of these two biomarkers. Furthermore, these markers were evaluated in patients who, like in all studies on the subject, do not necessarily have a diagnostic issue. Ideally, only patients facing a diagnostic challenge for their PJI should be included.

In conclusion, we present an algorithm for routine PJI diagnosis using synovial fluid biomarkers (Figure 4). A cut-off value of 80 mg/L for synovial calprotectin significantly enhances the test's performance, achieving a sensitivity of 94.2% and a specificity of 84.2%. Furthermore, this cut-off value reduces the number of false positives compared to a threshold of 50 mg/L. When applied to our cohort, this diagnostic algorithm demonstrates excellent diagnostic performance, with a sensitivity of 95.7%, a specificity of 95.5%, a negative predictive value (NPV) of 95.5%, and a positive predictive value (PPV) of 95.7%. Only two patients were classified as false positives and two as false negatives (Figure S1). This proposed diagnostic algorithm using synovial fluid biomarkers provides an accurate and reliable tool for the routine diagnosis of PJIs, with the potential to enhance clinical decision-making and patient outcomes. Even if these results are very interesting, they should be interpreted with caution due to the limited sample size and should be confirmed in larger cohorts or registry-based studies.

CRediT authorship contribution statement

**L. Ruffier d'Epenoux:** Writing – review & editing, Writing – original draft, Validation, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. **M. Robert:** Writing – original draft, Investigation, Formal analysis. **R. Lecomte:** Writing – original draft, Validation. **C. Nich:** Writing – original draft, Validation. **P. Bémer:** Writing – review & editing, Writing – original draft, Validation, Methodology, Formal analysis, Conceptualization. **S. Corvec:** Writing – review & editing, Writing – original draft, Validation, Methodology, Formal analysis, Conceptualization.

Acknowledgments

We would like to thank the lab technicians (Sonia Golfier, Karima Zouba) and the medical residents for their contribution to this study.

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Appendix

<figure><img src="image_9.png" /><figcaption>**Figure S1.** Proposed algorithm tailored to our study cohort. PJI, periprosthetic joint infection; sCalpro, synovial calprotectin; sCRP, synovial CRP; sLE, synovial leukocyte esterase; CRP, serum C-reactive protein; sWB, synovial white blood cell count; MSIS, Musculoskeletal Infection Society.</figcaption></figure>