The role of newer synovial biomarkers in the diagnosis of periprosthetic infections

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1 Page 1 of 6 Diagnosis & Treatment The role of newer synovial biomarkers in the diagnosis of periprosthetic infections VK Viswanathan 1 *, P Nayak 2 Abstract Introduction Periprosthetic joint infection has remained the most annihilating adversity and intriguing challenge confronting orthopaedic surgeons following joint arthroplasty procedures globally. The criticality of a surgeon s decision in identifying an underlying infection prior to revision arthroplasties and in managing such situations appropriately can never be understated. The current article reviews the role of novel synovial fluid biomarkers, including Polymerase Chain Reaction, C-Reactive Protein, leukocyte enzymes and other multiple molecular markers in identifying early PJIs. Conclusion The current literature is extremely scarce on these newer modalities, and their role in standard clinical practice is still greatly dubious and debatable. The research in this subject has a long way ahead and is reckoned in future, to elucidate our perspective and outlook on such scenarios. Introduction Periprosthetic joint infection (PJI) has remained the most annihilating adversity and intriguing challenge confronting orthopaedic surgeons following joint arthroplasty procedures globally. The criticality of a surgeon s decision in identifying an underlying infection prior to revision arthroplasties and in managing such *Corresponding author drvibu007@gmail.com 1 Sancheti Institute of Orthopedics and Rehabilitation, Shivaji Nagar, Pune, Maharashtra PD Hinduja Hospital, Mahim West, cadell road, Veer Savarkar Marg, Mumbai, situations appropriately can never be understated. Despite the advent of multitudinous, advanced and more accurate diagnostic modalities in describing subclinical infectious processes 1 6, there still exist certain perplexing circumstances where the surgeon is faced with ambiguous and equivocal investigation results 7. Novel and simpler diagnostic tests have been debated and investigated upon lately worldwide, which may aid orthopaedists by providing additional evidences supporting the presence or absence of infections in such situations of dubiety. The role of diverse synovial fluid biomarkers in early detection of PJIs has recently been the subject of interest among investigators. Apart from the bacterial detection techniques (including bacterial culture 8 or other genetic detection procedures like PCR 9 10 ), modalities that involve direct detection of the joints response to these septic stressors have gained immense attention. These include the inflammatory, acute phase markers including CRP 11 15, leukocyte enzymes 16 and multiple cytokines and molecular markers 17 from the aspirated joint fluid. These, in addition to the total and differential counts of recruited synovial white blood cells, may develop into potentially invaluable tools in PJI diagnosis. The current review article purports to focus upon these aforementioned newer investigative techniques, which may form an important part of pre-operative work-up for revision arthroplasties and may aid in probing into the existence of any probable, underlying infections. Discussion The gold-standard technique to diagnose periprosthetic joint infections has long eluded the medical fraternity. Parvizi et al. 18 had proposed the following diagnostic criteria to identify this dreaded complication: y Positive sinus tract communicating with the prosthesis or y Pathogen isolated by culture from two or more separate tissues or fluid samples from the affected prosthetic joint or y When four of the following six criteria exist: y Elevated serum Erythrocyte Sedimentation Rate (ESR)and C-Reaction Protein (CRP) y Elevated synovial white blood cell count y Elevated synovial polymorphonuclear percentage y Presence of purulence in the joint y Isolation of a microorganism in one culture of peri-prosthetic tissue or fluid y Greater than five neutrophils per high-power field in five high-power fields observed from histologic analysis of periprosthetic tissue at 400 magnification. Joint aspiration is an important investigation and is carried out as indicated by the guidelines proposed by AAOS (American Acedemy of Orthopedic Surgeons) on the basis of clinical probability of infection and results of serum ESR and CRP 19. Apart from all the above routine investigations of the synovial fluid, various adjunctive modern laboratory investigations may be employed in individual situations, as necessary.

2 Page 2 of 6 Pathophysiology of prosthetic infections The most important pathophysiology 20 of prosthesis-related infections includes adhesion of the bacteria to the surface of the implant. This process may be aided by the surrounding biomaterials and adjoining joint fluid and involves two distinctive phases, namely the nonspecific reversible phase and the specific irreversible phase. The most common bacteria causing prosthetic infections are the Gram-positive organisms, especially the Staphylococci, and these bacteria have a relatively specific mode of intracellular internalisation that augments their infective capability and resistive potential to antibiotics. Techniques to improve yield in bacterial detection methods Molecular diagnostic techniques to detect bacterial infections have been extensively studied in the last few years. Bergin et al. 21 proposed the role of RTPCR in detecting bacterial rrna, which has a sensitivity equivalent to intraoperative cultures as well as 100% specificity and positive predictive value. The principal advantage of this technique over culture is the possibility of bacterial detection even after the inception of antibiotic therapy. Jacovides et al. 22 had employed the sophisticated Ibis Biosciences T5000 biosensor system, which uses pan-domain primers in a series of PCRs to identify all bacteria and fungi. Synovial fluid was collected from 82 patients prospectively undergoing 87 replacement procedures. Both culture and Ibis analysis were carried out. Apart from identifying the pathogen in all cases that were grown on cultures, this novel modality also detected organisms in four out of five culture-negative cases. Achermann et al. 23 prospectively studied 37 prostheses that were removed following a diagnosis of PJI. The implants were sonicated, and the resultant fluid was cultured and sent for multiplex PCR. They could identify infection in all the 19 patients (100%) using multiplex PCR despite being on antibiotics, while sonication cultures grew the organism in eight such patients only (42%). Detecting bacterial nucleic acids in synovial fluid can be technically complicated due to the presence of inhibitors in the specimen Various techniques had been advocated and practiced by investigators to overcome this difficulty and provide appropriate sample preparation. Kathju et al. 28 had described a technique involving a single round of multiple displacement amplification on purified nucleic acids, so as to concentrate the template DNA. Although the PCR analysis of the tissues may be considered an ingenious method to diagnose PJI, there have been issues raised regarding the possible false-positive results secondary to tissue contamination problems. Bjerkan et al. developed and designed a specific way of performing real-time 16S rrna gene PCR analysis, which is purported to minimise the chances of false positivity and obviate this problem. Detection of synovial CRP in the diagnosis After an extensive search of the literature, we could identify seven articles that had discussed the diagnostic utility of CRP in the aspirated joint fluid in PJI. Vanderstappen et al. 15 had included a total of 43 patients and compared the roles of serum (from 24 serum samples collected) and synovial CRP levels (from 44 synovial fluid samples) in the diagnosis of infection. Parvizi et al. 29 had studied 66 patients undergoing revision total knee arthroplasty, who were classified as infected or not based on standardised criteria. Synovial CRPs were measured using an individual CRP assay (15 samples: 10 infected and 5 non-infected) and multiplex immunoassay platform (59 samples: 25 infected and 34 non-infected). Another study by Parvizi et al. 30 included prospective collection of synovial fluid samples in 63 patients undergoing primary or secondary replacement surgeries. Forty-three of these patients were classified into the aseptic group, while the remaining was considered septic. Cipriano et al. 31 studied 803 patients undergoing 871 consecutive hip and knee arthroplasties (810 of these patients had non-inflammatory arthritis and the remaining had inflammatory joint pathologies). Cashman et al. 32 had prospectively collected 50 synovial samples from patients undergoing knee replacements and had divided them into three groups: primary Total Knee Arthroplasties (TKAs), infected arthroplasties and the non-infected ones. All samples were assessed for CRP, cell count and differential count. Another study 33 involved 53 patients undergoing TKA, and synovial fluid CRP levels were measured using individual ELISA and multiplex immunoassay platform. Parvizi et al. 34, in another article on the same subject, discussed the CRP levels in 72 patients, in whom joint fluid analysis was carried out just before revision total knee arthroplasties. Twentynine of these knees were infected, and all the samples were run on multiplex immunoassay platform. Fifteen other samples (of which 10 were infected) were analysed using ELISA kit. The results of all these studies have been shown below (Table 1). Thus, most of the studies (although very limited) have confirmed the role of synovial fluid CRP in PJI. The need for a large-scale trial is, however, warranted in the current scenario to analyse its necessity in routine synovial studies and incorporation into management protocol. Detection of synovial leukocyte esterase in the diagnosis Based on the hypothesis and assumption 16,35 that infected synovial fluid is flooded with leukocytes and the measurement of an enzyme secreted

3 Page 3 of 6 Table 1 of studies discussing role of synovial CRP in PJI diagnosis S.No. Study Results Observations 1 Vanderstappen et al Area Under Curve (intra-articular CRP) AUC (serum CRP) 2. Cutoff points for intra-articular CRP 1.8 mg/l and 2.8 mg/l 2 Parvizi et al Synovial CRP differed between infected and uninfected joints in the multiplex and serum analyses. 2. AUC 0.84 (individual assay) AUC 0.91 (multiplex assay) AUC 0.88 (serum CRP) 3. A. ELISA assay: 70% sensitive, 100% specific B. Multiplex assay: 84% sensitive, 97.1% specific C. Serum CRP: 76% sensitive, 93.3% specific 3 Parvizi et al Difference in mean synovial CRP between the septic cohort (40 mg/l) vs. aseptic failure (a mean of 2 mg/l) (p < ) 2. Sensitivity 85% Specificity 95% (at threshold of 9.5 mg/l) 3. AUC Cipriano et al. 31 All synovial and serum-based tests equally useful in inflammatory and non-inflammatory arthritis 5 Cashman et al. 32 Synovial fluid CRP: 1. Native knees: 0.3 ± 0.4 (p < 0.006) 2. Aseptic knees: 0.2 ± 0.5 (0.001) 3. Infected knee arthroplasties: 4.3 ± 4.1 (significantly higher values) At synovial CRP cutoff of 1, for a diagnosis of infection: Sensitivity 89% Specificity 100% 6 Parvizi et al. 33 AUC: 1. Individual ELISA analysis: Multipex assay: Serum CRP assay: Sensitivity and specificity: % and 87.5% for the individual ELISA % and 88.9% for the multiplex assay 3. 80% and 89.7% for the serum CRP assay 7 Parvizi et al. 34 (Interim report) 1. RBM analysis of synovial CRP: showed threshold level of mg/l with: Sensitivity 87.1% Specificity 97.7% Positive Predictive Value 96.4% Negative Predictive Value 91.5% Accuracy 93.3% 2. Mean aseptic synovial fluid CRP value was 1.64 mg/l while the mean septic synovial fluid CRP value was mg/l (p < ). 3. Receptor Operated Characteristic curve showed 84.8% sensitivity 95.5% specificity at a CRP synovial fluid threshold of 9.5 mg/l 4. Statistically significant differences between the groups for serum CRP, synovial fluid CRP, ESR, synovial WBC count and neutrophil differential count (p < 0.001). 1. Both serum and synovial CRPs showed very high diagnostic strengths 2. No significant difference in strengths between the two parameters Synovial CRP more accurate than serum CRP Synovial CRP promising marker Synovial CRP low-cost, potential tool Synovial CRP more accurate than serum CRP CRP concentrations in the synovial fluid rather than serum improve the diagnostic accuracy of this test in identifying PJI

4 Page 4 of 6 Table 2 of studies discussing role of synovial Leukocyte Esterase in PJI diagnosis S No. Study Results Conclusion 1 Parvizi et al Based on clinical, serological and operative criteria 30 of the 108 knees infected 78 uninfected by the same cells by colourimetric techniques may serve as a simple, yet invaluable and specific ancillary investigative modality, investigators began their ground-breaking work on this subject. This technique, although had seldom been employed previously in orthopaedic problems, had worked consistently in diagnosing infections elsewhere in body fluids, including urinary tract, pleural effusion, sputum, bronchoalveolar lavage, middle ear effusion, peritoneal effluent and gastric mucosa. Parvizi et al. 16 had studied during the period May 2007 April 2010 a total of 108 synovial fluid samples (preoperative samples in suspected joint infections and intraoperative 2. With only a ++ reading as the cutoff, Sensitivity 80.6% (95%CI 61.9% to 91.9%) Specificity 100% (95%CI 94.5% to 100.0%) Positive predictive value 100% (95%CI 83.4% to 100.0%) Negative predictive value 93.3% (95%CI, 85.4% to 97.2%) 3. Strong correlation with A. Percentage of Polymorphonuclear leukocytes (r = ) and B. Total white blood cell count (r = ) in the aspirate A. ESR (r = ) and B. C-reactive protein (r = ) in serum 2 Wetters et al LER strips-positive (23.3%) 106-negative (47.5%) 65 strips (29.2%) unable to be read secondary to debris or blood 2. With synovial fluid WBC >3000 per microlitre as an indicator of PJI: Sensitivity 92.9% Specificity 88.8% 3. With positive cultures for diagnosis of PJI: Sensitivity 93.3% Specificity 77.0% 4. Where reoperation was performed and a combination of factors were used for the diagnosis: Sensitivity 100% Specificity 86.8% samples in those undergoing revision replacements). The aspirate was examined for leukocyte esterase enzyme using colourimetric strips, and the colour changes were graded as negative, trace, + or ++. Wetters et al in 2012 had included a total of 223 consecutive total hip or knee arthroplasties, which were evaluated for PJI using leukocyte esterase reagent (LER) strips. In another study in 2011, a total of 223 patients who were suspected to have developed PJI (180 TKAs and 43 Total Hip Arthroplasties (THAs)), testing with LER strips was done in the office (156 patients) or in the operating room (67 patients). Researchers only considered LER strips positive for infection, 1. Simple 2. Inexpensive 3. Both rules out and confirms infection 4. Valuable addition to surgeon s armamentarium 5. Additional multicentric trials required 1. Rapid 2. Inexpensive 3. Sensitive 4. Utility is limited, however, by blood or debris in the synovial fluid making them unreadable in 33% of cases if the test was reported for moderate or large reactivity (+ or ++). The results of these studies have been shown below (Table 2). Detection of other synovial biomarkers in the diagnosis Recently, researchers have assayed to include a set of test detecting levels of multiple cytokines or molecular markers in the synovial fluid aspirate. These markers may be anticipated to represent more specific tools in aiding the discovery of early infections, and thus there is considerable enthusiasm regarding their future roles. The role of serum IL6 17 and other molecular proteins has been discussed by researchers

5 Page 5 of 6 for long. Deirmengian et al. 7 had prospectively collected synovial fluid from 14 patients with suspected infections, and 37 patients suspected to have an aseptic failure of their implants. The synovial fluid was tested for 23 potential biomarkers. Among them, 12 synovial biomarkers had higher mean levels in infected synovial fluids as against aseptic joints. Synovial fluid levels of IL1 were observed to have a relative increase of around 258 times in infected prostheses, in comparison with the aseptic ones. IL1 and IL6 levels in the joint fluid accurately classified all patients with sensitivity, specificity, positive predictive value, negative predictive value, and accuracy approaching one. In a study by Jacovides et al. 37, 74 synovial samples (31 infected and 43 uninfected) were analysed. Proteomic analysis and Receiver Operating Characteristic curve analyses were conducted on 46 proteins. Of these, IL6, IL8, α2 macroglobulin, CRP and vascular endothelial growth factor had area under curve of greater than 0.9. The authors have expressed their enthusiasm regarding the prospective, indispensable role of these proteins in this field. Conclusion The issue of peri-prosthetic infections has flummoxed the orthopaedic fraternity for ages, and the goldstandard management that can be universally employed has not been determined hitherto. Synovial aspirates provide the most direct evidence for intra-articular pathologies and infections, and the immediate markers for local, inflammatory activity and products of inflammatory cascade within the joint, heretofore discussed, can be useful indicators of these joint insults. They may act as additional components of synovial fluid testing (apart from the routinely performed smear analysis, total and differential leukocyte counts and bacterial culture). 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