➢ Molecular methods that involve the use of technology such as polymerase chain reaction to amplify existing bacteria can increase the likelihood of identifying the pathogen responsible for periprosthetic joint infection.
➢ Synovial biomarkers, such as C-reactive protein (CRP), leukocyte esterase, α-defensin, human β-defensin-2 (HBD-2) and HBD-3, and cathelicidin LL-37 are elevated in patients with periprosthetic joint infection and may be valuable markers for diagnosing such an infection with use of point-of-care devices.
➢ Serum biomarkers, such as interleukin (IL)-4, IL-6, procalcitonin, and soluble intercellular adhesion molecule-1 (sICAM-1), have been shown to be elevated in patients with periprosthetic joint infection and may provide markers for diagnosing such an infection and for determining the proper time to perform reimplantation.
The diagnosis of periprosthetic joint infection remains challenging and relies on a combination of clinical history, physical examination findings, and laboratory tests. The Musculoskeletal Infection Society (MSIS) convened a panel of experts to develop criteria for diagnosing periprosthetic joint infection1. The criteria developed by the workgroup currently represent the most commonly used definition of periprosthetic joint infection. The workgroup identified two major criteria that were indicative of periprosthetic joint infection: (1) the presence of a communicating sinus tract with the prosthesis or (2) the isolation of a pathogen from two different tissue or fluid samples from the joint. In addition, the workgroup identified six minor criteria and indicated that diagnosis of periprosthetic joint infection was very likely when four of the following six criteria were present: (1) an elevated erythrocyte sedimentation rate (ESR) or C-reactive protein (CRP) level2, (2) an elevated synovial white blood-cell (WBC) count, (3) an elevated level of synovial polymorphonucleocytes (PMNs), (4) the presence of purulence, (5) the isolation of one pathogen in tissue or fluid, or (6) more than five neutrophils per high-powered field in tissue within the affected joint when viewed at 400× magnification. Recently, an International Consensus Group on Periprosthetic Joint Infection endorsed the MSIS criteria for periprosthetic joint infection and modified them slightly3. The MSIS group provided a stepwise approach for the diagnosis of periprosthetic joint infection4.
The methods of diagnosing periprosthetic joint infection have been confined to traditional methods of determining infection, including measurements of the levels of serological inflammatory markers, the synovial cell count, and the neutrophil differential as well as conventional culture. However, these tests have major deficiencies; on many occasions, for example, the infecting organism may not be isolated on culture or the serological markers may not be elevated in cases of innocuous infections with organisms like Propionibacterium acnes5. Because of the limitations of these traditional tests for the diagnosis of periprosthetic joint infection, some authorities believe that a considerable number of aseptic failures are in fact due to infections that either have escaped diagnosis or have not been adequately investigated. In one study, 10.5% (thirty-four) of 323 so-called aseptic failures were believed to have occurred as a result of periprosthetic joint infection6. Another major limitation of the available tests is that they were not specifically developed for the purpose of diagnosing periprosthetic joint infection, and hence the optimal threshold for a positive test for periprosthetic joint infection has not been established.
These limitations have compelled the orthopaedic community to seek alternative and more advanced strategies for the diagnosis of periprosthetic joint infection. This article will outline some of the recent and emerging technologies that hold promise for the diagnosis of periprosthetic joint infection.
Molecular Detection Methods
Microbiological culture routinely has been used for the diagnosis of periprosthetic joint infection. Traditional culture, despite its established value, has many limitations: isolation of an organism on culture may be difficult to determine if previous providers have already administered antibiotics, the process usually takes a few days, a false-negative result may occur if the culture is not sensitive enough to detect an organism, and a false-positive result may occur if the sample is contaminated during transport and processing. Therefore, there is increasing interest in the use of molecular genetics to improve the diagnosis of infection by reducing the time to diagnosis and potentially reducing the number of false-negative results.
The benefit of molecular methods, such as polymerase chain reaction, is that any small amount of bacterial DNA that is present within a synovial fluid or tissue sample can be amplified, leading to identification of the infecting organism7,8. The main limitation of polymerase chain reaction relates to its extreme sensitivity and increased rate of false-positive results as dead or noninfectious bacteria also can be amplified. In recent years, efforts have been made to improve the specificity of polymerase chain reaction by measuring 16S ribosomal RNA (rRNA). The intention is that targeting rRNA will result in amplification of only the genetic material of live bacteria, thus reducing the number of false-positive results9. In addition, improving polymerase chain reaction analysis by using specific DNA targets, such as the mecA gene for identifying methicillin-resistant Staphylococcus aureus (MRSA), also can reduce the rate of false-positive results.
Another methodology to reduce the number of false-positive results with use of conventional polymerase chain reaction relates to the use of multiplex polymerase chain reaction. In recent years, commercial instruments that utilize a more sophisticated methodology of genetic material amplification for the detection of infection have been introduced. The IBIS T5000 Biosensor (Abbott Laboratories, Abbott Park, Illinois) is a machine that uses broad-range polymerase chain reaction and multiple pairs of species-specific primers to identify bacteria, viruses, fungi, and protozoa10. Electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) is performed, and the signals are processed and analyzed for base composition. These results are compared with a database of microbes to identify the correct pathogen, including all bacteria; the fungus Candida; vancomycin-resistant markers in enterococci, including vanA and vanB; carbapenem-resistant gram-negative organisms; and methicillin-resistant staphylococci. Studies have demonstrated that the IBIS T5000 is effective for diagnosing periprosthetic joint infection, particularly in cases in which the culture results are negative. Although less of an issue than it is for conventional polymerase chain reaction, extreme sensitivity continues to affect this technique; an infecting organism was detected in 87.7% (fifty) of fifty-seven culture-negative cases in one study and in 26.2% (seventeen) of sixty-five aseptic revision cases in another study11,12.
A newer organism-identification system, namely, PLEX-ID (Abbott Laboratories), was introduced recently. Similar to the IBIS T5000, PLEX-ID uses polymerase chain reaction and ESI-TOF-MS to identify organisms. PLEX-ID offers broad identification of as many as 3100 species of organisms, including influenza13, highly pathogenic bacteria14, and fungi15. To our knowledge, no studies have been performed to evaluate the role of this system in diagnosing periprosthetic joint infection. However, it is believed that the ability of this system to perform high-resolution subtyping and detection of drug resistance may hold promise for orthopaedics.
While the IBIS T5000 and PLEX-ID have increased the detection of organisms, the major cost of these machines may prohibit many centers from having them available. In addition, the results of the tests may not be available for as long as forty-eight hours, as processing often is performed from a centralized location to reduce costs. Another type of molecular technique, namely, isothermal recombinase polymerase amplification (RPA), also has been utilized to help to detect the presence of infecting organisms. TwistDx (TwistDx Limited, Babraham, Cambridge, England) is a point-of-care device that is portable and can be used to perform rapid identification of organisms within ten to fifteen minutes with use of RPA16. Recombinases pair oligonucleotide primers with sequences on duplex DNA, which activates DNA synthesis and amplification at room temperature. This device is highly sensitive for detecting single molecules, such as MRSA, Salmonella, and other organisms with the appropriate primers but is unable to detect multiple organisms at one time.
Another method of detecting the presence of bacterial DNA involves the use of microbead-detector technology17. In this model, a reporter molecule is attached to a peptide substrate that is subsequently covalently attached to a microbead anchor. The reporter molecule is placed in the form of a patch, and if the microbead detector is exposed to specific DNA, the color indicator is activated, and the presence of a pathogen is confirmed. It is believed that further refinements in this technique may allow it to be used to screen patients for MRSA infection or even for the diagnosis of skin-based and deep organ infections.
Synovial Fluid Biomarkers
The era of biomarker development for the diagnosis of numerous conditions is upon us. The field of orthopaedics stands to benefit from this technology, particularly in terms of the ability to diagnose periprosthetic joint infection. The utility of biomarkers for the diagnosis of periprosthetic joint infection has been explored over the last few years18. A number of potential synovial fluid biomarkers that are indicative of periprosthetic joint infection also have been identified (Table I). Synovial fluid biomarkers can broadly be divided into cytokines and biomarkers with antimicrobial functions19. Cytokines, such as interleukin (IL)-1β, IL-6, IL-8, and IL-17, are released from macrophages at the site of infection and are elevated in the synovial fluid of patients with periprosthetic joint infection compared with the levels in patients undergoing revision for reasons other than infection20. Other cytokines, such as tumor necrosis factor-α (TNF-α) and interferon-δ (IFN-δ), are also elevated in patients with periprosthetic joint infection21. Finally, vascular endothelial growth factor (VEGF), which is stimulated in angiogenesis, is found in increased levels in the synovial fluid of patients with periprosthetic joint infection18. Although all of these cytokines are elevated in patients with periprosthetic joint infection, the problem with using them as biomarkers is that they are also often indiscriminately elevated in association with other inflammatory conditions, such as rheumatoid arthritis, inflammatory bowel disease, sarcoidosis, and vasculitis. Thus, the search for a more specific biomarker for periprosthetic joint infection continues.
Efforts have been made to seek a biomarker that is induced in the presence of an infecting organism. These antimicrobial biomarkers are assumed to have high specificity for periprosthetic joint infection while retaining the sensitivity of a general biomarker for detecting infection22. Some of the potential antimicrobial biomarkers include synovial CRP, leukocyte esterase, α-defensin, human β-defensin-2 (HBD-2) and HBD-3, and cathelicidin LL-37. CRP is a protein synthesized in the liver that is released when increased macrophages are present during acute inflammation. While serum CRP is currently a part of the algorithm for diagnosing periprosthetic joint infection, synovial fluid CRP has been reported to hold better promise for the diagnosis of periprosthetic joint infection, as it appears to be more specific23. Clinical tests comparing synovial fluid from patients with and without infection demonstrated that the sensitivity of the test was 85% and the specificity was 95% at a threshold of 9.5 mg/L24. With an area under the curve (AUC) of 0.92, synovial fluid CRP is a useful diagnostic tool for determining the presence of periprosthetic joint infection. The limitation of this test relates to the unwillingness of some hospital laboratories to measure the level of CRP in the synovial fluid, as the available machines have not been calibrated for this purpose.
Another biomarker with antimicrobial function is leukocyte esterase, an enzyme secreted by neutrophils in response to the presence of infecting organisms. The measurement of leukocyte esterase in the urine with use of a dipstick technique has been used for decades. The utility of this dipstick test for the diagnosis of periprosthetic joint infection was recently explored25. This rapid and inexpensive test appeared to carry a sensitivity of 93.3% and a specificity of 77.0% for the diagnosis of periprosthetic joint infection when isolation of an infecting organism on culture was used as the “gold standard” for diagnosing periprosthetic joint infection. The main limitation of the dipstick leukocyte esterase test relates to its inability to be used for patients with bloody synovial fluid, which is seen in as many as 30% of cases, as the color of blood interferes with the interpretation of this test, which depends on color changes in the fluid26.
Very recently, another promising synovial biomarker has been introduced. Human neutrophil protein (HNP), or α-defensin, has been found to hold great promise for the diagnosis of periprosthetic joint infection. HNPs are peptides that are released from neutrophils in response to the presence of bacteria. A preliminary study evaluating HNP1-3 as a potential marker for diagnosis of periprosthetic joint infection demonstrated a sensitivity of 97% and a specificity of 96% at a cutoff value of 5.2 µg/mL27. Furthermore, α-defensin exhibited no overlap of values between patients undergoing revision because of infection and those undergoing revision for other reasons, making it an ideal molecular test for the diagnosis of periprosthetic joint infection.
HBD-2 and HBD-3 are similar to α-defensin in that they are secreted by neutrophils and epithelial cells in response to inflammation. HBD-2 and HBD-3 exhibit antimicrobial activity against Candida and gram-negative organisms. HBD-3 was found to be significantly elevated in the synovial fluid aspirates of patients with periprosthetic joint infection (AUC = 0.745, p = 0.014), whereas HBD-2 was elevated in the serum of patients with periprosthetic joint infection (AUC = 0.738, p = 0.162)21.
LL-37 is a cationic antimicrobial protein peptide that is a member of the cathelicidin family. It exerts multiple biological effects, including the prevention of biofilm formation, the induction of immune mediators, including IL-8, and the regulation of the inflammatory response28,29. In the study by Gollwitzer et al., LL-37 was found to be elevated in the synovial fluid of patients with periprosthetic joint infection, with an AUC of 0.875, a sensitivity of 80%, and a specificity of 85% for the diagnosis of periprosthetic joint infection21.
The inability to obtain synovial fluid in some cases, particularly those in which a spacer is in place, and the invasiveness of joint aspiration have led some authorities to seek other methods for diagnosing periprosthetic joint infection, such as percutaneous biopsy, which is invasive, or testing for serum biomarkers, which is less invasive30,31. Not only do serum biomarkers hold promise for the diagnosis of periprosthetic joint infection, they also may be used to determine the resolution of periprosthetic joint infection and to aid in the identification of the optimal timing for reimplantation during a two-stage exchange arthroplasty. Although ESR and CRP are the most commonly used serum tests for the diagnosis of periprosthetic joint infection, these tests are nonspecific, as their results are elevated in association with inflammatory conditions. Similar to the biomarkers found in synovial fluid, some proinflammatory cytokines, such as IL and TNF-α, have been found to be elevated in the serum of patients with periprosthetic joint infection (Table II)21. Di Cesare et al. reported that IL-6 was elevated (>10 pg/dL) in patients with periprosthetic joint infection and had a sensitivity of 100% and a specificity of 95% for the diagnosis of periprosthetic joint infection32. This finding was confirmed when the serum levels of both IL-4 (AUC = 0.745; sensitivity, 60%; specificity, 90%; p < 0.001) and IL-6 (AUC = 0.687; sensitivity, 47%; specificity, 95%; p = 0.002) were found to be significantly elevated in patients with staphylococcal periprosthetic joint infection as compared with the serum levels in patients with aseptic failures21. The serum level of TNF-α (>40 ng/mL), despite its low sensitivity (43%), also has been found to be promising for the diagnosis of periprosthetic joint infection with a high specificity (94%) and a relatively high accuracy (83%)33. Another promising marker for the diagnosis of periprosthetic joint infection is soluble intercellular adhesion molecule-1 (sICAM-1), which has been shown to be elevated in patients with inflammatory conditions such as atherosclerosis, in which these molecules are activated when there is damage to the endothelium34. sICAM-1 has been shown to be significantly elevated in patients with periprosthetic joint infection as compared with patients undergoing revision because of aseptic loosening (p = 0.0002)35,36.
In addition to proinflammatory cytokines, peptide precursors like procalcitonin (PCT) are elevated in the presence of proinflammatory stimuli like bacteria. PCT has been found to be elevated in patients with sepsis37 and those with septic arthritis38, even when compared with the serum levels in patients with inflammatory arthritis39. One study showed that elevation of PCT to a level of >0.35 ng/mL had a sensitivity of 80% and a specificity of 37% for the diagnosis of periprosthetic joint infection40. However, the utility of PCT as a potential serum marker for the diagnosis of periprosthetic joint infection was questioned in another study in which PCT (>0.3 ng/mL) demonstrated a high specificity (98%) but a low sensitivity (33%)33. Other studies have demonstrated no significant difference in the level of serum PCT between patients with and without periprosthetic joint infection35,36. These findings raise the possibility that there may be a wide temporal variation in the serum level of some markers, such as PCT. Thus, the search for the optimal serum marker that can be used for the diagnosis of periprosthetic joint infection and can provide information regarding the resolution of infection and the optimal timing of reimplantation continues. The sensitivity and specificity of serum and synovial fluid biomarkers are shown in Table III. An ongoing multicenter study is being performed to evaluate the role of some potential serum biomarkers in diagnosing periprosthetic joint infection and in determining the optimal timing of reimplantation.
The diagnosis of periprosthetic joint infection continues to pose a challenge to the orthopaedic community. With recent advances in molecular biology and the development of promising biomarkers for the diagnosis of various conditions, it is hoped that molecular techniques will replace conventional methods that continue to show low accuracy for the diagnosis of periprosthetic joint infection. In addition, continued developments in the areas of genomics and metabolomics across the entire medical field are likely to benefit orthopaedics in general and the treatment of periprosthetic joint infection in particular.
Source of Funding: No external funds were received in support of this study. Ongoing research at our institution is seeking a serum biomarker for the diagnosis of periprosthetic joint infection and is being funded by the Orthopaedic Research and Education Foundation (OREF).
Investigation performed at the Rothman Institute, Philadelphia, Pennsylvania
Disclosure: None of the authors received payments or services, either directly or indirectly (i.e., via his or her institution), from a third party in support of any aspect of this work. One or more of the authors, or his or her institution, has had a financial relationship, in the thirty-six months prior to submission of this work, with an entity in the biomedical arena that could be perceived to influence or have the potential to influence what is written in this work. No author has had any other relationships, or has engaged in any other activities, that could be perceived to influence or have the potential to influence what is written in this work. The complete Disclosures of Potential Conflicts of Interest submitted by authors are always provided with the online version of the article.
- Copyright © 2014 by The Journal of Bone and Joint Surgery, Incorporated