Periprosthetic joint infection (PJI) is a serious and devastating complication that may occur after native joint replacement with major health and economic consequences. Indeed, implant failure can necessitate additional surgery with substantial higher costs for the health system and a significant reduction of patient quality of life . Considering that the health and economic PJI burden is estimated to increase in the next 20 years due to the aging population and the prevalence of comorbidities, an accurate and objective recognition of these aforementioned infections is fundamental to guarantee appropriate treatment to patients with PJIs, thus avoiding unnecessary antibiotic therapies for individuals with aseptic failure .
To date one of the main problems in the management of PJIs is that the diagnosis of this kind of infection is confusing and often difficult to carry out. Complicating factors include the pathogenicity and virulence of the specific microorganism involved in the infection, the time of infection, the patient’s state of health and the type of joint involved .
In recent years, numerous efforts have been made to improve the diagnosis of PJIs, resulting in a multidisciplinary definition which takes into consideration different laboratory tests, tissue histology, biochemical and microbiological analysis, as well as clinical findings . These tests have facilitated PJI diagnosis, by evaluating, for example, the presence and the amount of specific biomarkers in the synovial fluid , , , , , . High amounts of cytokines and proteins with antibacterial activity have been detected in the synovial fluid of patients with an infected joint prosthesis , . α-Defensin is an antimicrobial peptide which is commonly secreted in synovial fluid in response to microbial products or pro-inflammatory cytokines. This molecule is then integrated into the cell membrane of the pathogen causing cellular death . The aim of the present work was to review current studies on α-defensin in the diagnosis of PJIs as an early synovial marker of infection which, today, is proposed directly to physicians as a point-of-care test (POCT). Consequently, the present paper can be considered a warning for readers about all those tests which completely bypass the analysis laboratory, representing a serious confounding factor for surgical decisions.
α-Defensin as marker of PJIs
Defensins are endogenous, microbicidal peptides active against enveloped viruses, fungi and both Gram-negative and Gram-positive bacteria . α-Defensins, in particular, are very abundant in neutrophils and in macrophage populations and are released primarily from polymorphonuclear cells in response to pathogens . The resulting death of pathogenic microorganisms is due to the ability of defensins to form “channel-like” pores in the cell membrane and/or bind and cover the microbial membrane leading to the disruption and lysis of microorganisms . Generally, α-defensin is measured in synovial fluid by means of an enzyme-linked immunosorbent assay (ELISA), which is optimized specifically for application to this kind of specimen; as a consequence, all effects of variable viscosity between analyzed samples are removed. The production of α-defensin is controlled by numerous pro-inflammatory cytokines, such as interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), which are able to upregulate α-defensin expression . More interestingly, α-defensin levels do not seem to be affected by antibiotic administration for the treatment of PJIs before diagnostic evaluation, as a decrease in α-defensin levels has not been observed after antimicrobial administration . Conversely, C-reactive protein, white blood cells and polymorphonuclear cells are all significantly reduced after antibiotic treatment and this can lead to confounding results and an incorrect diagnosis of PJI . Moreover, the α-defensin ELISA test has also been highlighted as having a high sensitivity and specificity (greater than 95%) for hip and knee PJI diagnosis . Furthermore, the synovial fluid α-defensin immunoassay was observed to be comparable to the test evaluating leucocyte esterase, an enzyme which is released from neutrophils in infected joint fluids . Positivity to leukocyte esterase is one of the minor criteria published by the Musculoskeletal Infection Society (MSIS) to define PJI, together with elevated serum C-reactive protein, elevated synovial fluid white blood cell count, elevated synovial fluid polymorphonuclear neutrophil percentage, positive histological analysis of periprosthetic tissue and single positive culture . The simplicity and greater effectiveness of the α-defensin immunoassay, compared with the more complex and potentially confounding MSIS criteria could make the former an attractive tool for the rapid and correct diagnosis of PJIs. Indeed, analysis of α-defensin levels, may be a useful tool to streamline the surgical approach, allowing the medical staff to immediately initiate an appropriate antibiotic therapy. However, Bonanzinga et al. demonstrated that although both positive and negative predictive values are high, only negative results may be considered as predictive in the diagnosis of PJI . If the α-defensin immunoassay is negative, it is very likely that no PJI is involved following the total hip arthroplasty (THA) and/or total knee arthroplasty (TKA); if the result is positive, instead, there is a high probability of PJI, but the elevated level of α-defensin may be due to reasons other than a periprosthetic infection. Metallosis, for instance, may predispose the α-defensin test to a false-positive result, being a misleading factor in interpreting α-defensin results and leading in turn to an incorrect diagnosis . Furthermore, Bingham et al. detected elevated levels of α-defensin, together with C-reactive protein (CRP), erythrocyte sedimentation rate (ESR) and cell count in two patients without PJI, hypothesizing that aseptic inflammation might be responsible for the elevated amount of α-defensin recorded in two patients , .
For this reason, the use of other specific PJI diagnostic criteria is of fundamental importance for the correct management of patients who undergo THA or TKA and/or with a suspected periprosthetic infection. The α-defensin immunoassay can be integrated with PJI microbiological and biochemical tests, as it provides robust and reliable results, but it should not be used as the only test that orthopedic surgeons and rehabilitation physicians can use to diagnose the presence of an ongoing periprosthetic infection.
Point-of-care testing (POCT) and α-defensin
POCT refers to the use of laboratory tests in proximity to patients. Generally, laboratory analysis by POCT are performed outside the laboratory, without sample preparation or pipetting steps, with ready-to-use reagents which do not require modification by the operator and with the immediate indications as to the potential therapeutic approach after the test results. Obviously, these analyses are very rapid and easy-to-interpret , , . Some specific POCT allow accurate and reliable results without delay, leading to a significant time advantage in vital decision making regarding diagnostic and therapeutic proceedings, with the subsequent improvement of patient outcomes.
In Europe, POCT devices are regulated under the 1998 European Directive 98/79/EC on in vitro diagnostic medical devices . In vitro diagnostic assays, including POCT, can generally be marketed if the product has passed a conformity assessment procedure and is awarded a CE mark, as this confirms conformity to European directives for in vitro diagnostic testing (IVD directive) . The test manufacturer is responsible for performing tests and and providing the device performance data, while the user is responsible for checking whether the POCT test is suitable for its diagnostic purpose and, consequently, useful for clinical application . However, to ensure the efficacy and regulatory compliance of POCT, different strategies have evolved in the last years. In numerous hospitals POCT programs are administered by representatives from nursing and laboratory staff and a medical director , , . Furthermore, correct POCT management calls for regular inspections by laboratories to verify regulatory compliance and promote a continuous provision of specialized staff in hospitals, various criteria for the activation of POCT exist (medical, economic and organizational aspects), but greater importance is given to turnaround time (TAT) . In particular, a moderate TAT is required for those parameters which provide specific information about vital functions and upon which the choice of therapeutic intervention may depend, especially in critical conditions . It is fundamental to keep in mind that the advent of POCT allowed the execution of high quality laboratory diagnostic tests by personnel without the necessary expertise in medical and laboratory technology. Consequently, the inexpert and uncritical use of POCT assays is not recommended and above all, these methods cannot substitute for the expertise and experience of a medical laboratory.
The function of POCT is to provide assistance in clinical decision-making, but to date there is not enough evidence to determine if these devices can improve patient management and reduce hospital admission . Moreover, the lack of specialized laboratory staff for the interpretation of analytical results may lead surgeons to formulate incorrect diagnosis, submitting patients to unnecessary surgery.
Today, a POCT assay (Synovasure, Zimmer, Warsaw, IN, USA) able to detect the presence of human α-defensin 1–3 in the synovial fluid of patients experiencing pain and/or inflammation in a replaced joint is directly proposed to orthopedic surgeons. Specifically, the “alpha-defensin flow later” test is a visual immunochromatographic assay composed of a single use device, a premeasured vial of dilution buffer, a disposable Microsafe tube and a sample cup . The synovial fluid is diluted appropriately and added to the test device where it migrates to the buffering pad and combines with a gold conjugate labeled with an anti-defensin antibody. Finally, the mixture migrates across the test line and the control line and after 10 min the result is provided to the operator . The device gives only two possible results:
positive: when the level of α-defensin is greater than the cut-off concentration, a test result line (“T”) will form on the device, together with a reddish-pink device control line (C-line);
negative: only a reddish-pink device control line appears on the device.
Interestingly, this α-defensin immunoassay provides consistent results which are related not to the type of microorganism involved in PJI, but instead it indicates only the presence of a “in-progress” infection , , , , . A recent study highlighted the broad spectrum of microorganisms responsible for the release of α-defensin in synovial fluid which, consequently, is detected by immunoassay . These results strengthen the diagnostic role that the α-defensin immunoassay may have in clinical practice even if to date, there are no data about the performance of the aforementioned test in the immediate postoperative period in severe immunocompromised patients or in subjects with severe inflammation which is not related to PJI. Moreover, Kasparek et al. underlined that the α-defensin intraoperative lateral flow test is equivalent to an MSIS-based diagnosis and is very useful to confirm the absence of PJIs . However, the authors evaluated the α-defensin POCT performance directly during surgery in the operating room, highlighting a sensitivity of 67% and a specificity of 93% . As expected, a perfect predictor would be described as 100% sensitive and specific, but of course any predictor is subject to a minimum error. For practical reasons, tests with sensitivity and specificity values above 90% have high credibility and consequently, data showed by Kasparek et al. underlined that a POCT assay with low sensitivity cannot be used alone for a correct PJI diagnosis. More interestingly, Frangiamore et al.  observed that an α-defensin POCT assay had a high sensitivity and specificity (>90%) for first-stage and single-stage revisions. However, the test performed less well when considering a second-stage revision; indeed, sensitivity was reduced to 67%, while specificity remained almost unchanged. Moreover, the authors did not perform the α-defensin test in the operating room directly during surgery but they collected the synovial fluid samples and sent them to an external laboratory for the analysis using the Synovasure test. Consequently, no reliable consideration about the in situ performance of the α-defensin POCT assay can be made.
To ensure a high quality and proficiency testing, for every ten POCT assays performed directly in the surgery room, one sample should be simultaneously tested and compared with the α-defensin ELISA in the central laboratory.
The main problem in using POCT tests is that many orthopedic surgeons have little experience and training in quality laboratory practices, often failing to appreciate the significance of quality control and proper documentation when using POCT assays .
Considering all these aspects, it is clear how the α-defensin assay, while representing a valid aid for orthopedics, should not be used as the only marker to rule out periprosthetic infections, as sometimes happens in clinical practice, but it should be integrated with the other MSIS criteria to obtain a more precise and accurate diagnosis.
Diagnosis of the different stages of PJI
When performing a PJI diagnosis, it is fundamental to consider the kind of infection affecting the patient; indeed, PJIs can be classified on the basis of symptom onset-time after the surgical implant.
Onset-time <4–6 weeks after surgery. Normally, these kind of infections are acquired during the surgery for microbial shedding on the operative field . More rarely, they can be due to the hematogenous spread from distal outbreaks not cleared correctly before the surgery . Early infections are often caused by highly pathogenic microorganisms, such as Staphylococcus aureus and Gram-negative bacteria, and if they are diagnosed early (<3 weeks) it is possible that bacterial colonization can still be limited. However, the only “time criterion” does not allow the formulation of a proper prognosis, as the outcome also depends on bacterial localization .
Onset-time between 3 and 24 months after surgery . They are considered to be acquired during surgery for microbial shedding on the operative field and are often caused by low-pathogenic microorganisms, such as coagulase-negative staphylococci, Enterococcus spp., Corynebacterium spp. and Propionibacterium acnes . As it is assumed that in these kind of infections the biofilm is well-structured, the most common therapeutic strategy is represented by a combined medical-surgical approach with the removal of arthroplasty, the cement positioning spacer, antibiotic therapy and repositioning arthroplasty after having eradicated the microbial infection .
Onset-time >24 months after surgery. These infections have a hematogenous pathogenesis from remote sites of infection. Debridement represents the surgical recourse which must be carried out as soon as possible, before the biofilm can become organized .
Considering these PJI classifications, it is clear that the diagnosis of acute infections is very easy to perform if compared to chronic and low-grade infections, and consequently, the α-defensin test can represent a useful tool to face acute infections. However, little information about its real efficacy in diagnosing chronic and low-grade infections exist, and to date there is no certainty that this test can discriminate between aseptic joint failure, low-grade infection and high-grade septic failure .
POCT is not a new phenomenon, ward glucose meters have been with us for decades, and remote blood gas analyzers have been used for nearly as long. Currently, results from POCT analyzers require careful checking and they present ongoing quality control dilemmas to both laboratory and regulatory staff . Generally, these systems require the manufacturer’s specific calibrators and QC materials in order to be used and checked by third parties. For any specific POCT, indeed, the ultimate goal would be to establish local and national regulatory standard procedures.
It is well demonstrated that the lack of accuracy of POCT measurements is related to the fact that POCT is generally performed by busy members of the clinical team and not by laboratory-trained individuals. Non-laboratory-trained individuals, indeed, often lack an understanding of the importance of quality control and quality assurance .
The α-defensin POCT assay may, of course, play an important role in PJI diagnosis, allowing the identification of a potential infection and reducing the time to intervention. At the same time, if this test is to be considered as true POCT assay and treated as a laboratory test, a comprehensive protocol which includes the entire testing process from the pre-analytical phase to the post-analytical phase for anyone performing testing must be provided and utilized. Validation of the test by laboratory professionals, evaluating the comparison and eventual accordance with the proposed immunoenzymometric assay for the same analyte, is mandatory for achieving the levels of effectiveness and accuracy needed for routine use. The test, used directly at the bedside or in outpatient services, could undoubtedly have value (i.e. outcome/costs), but the role of the clinical laboratory for defining pre-analytical, analytical and post-analytical quality is necessary, even for an adequate health technology appraisal. Such approaches should be mandatory to define not only the efficacy and effectiveness of the test, but also to evaluate a possible reimbursement from national and/or regional health services. The integration of knowledge from both laboratory professionals and clinicians is the real and current aim of pathology: a lack of cooperation will fail to assure the high quality and value of tests proposed by the market.
As with any laboratory test, as errors can occur at any point in the testing cycle, therefore improved quality control and assurance as well as personnel training should be ensured. For this reason, this test should not be used alone for diagnostic purposes, but rather it should be integrated with existing PJI criteria and combined with other specific and more appropriate tests on the basis of the stage of infection.
We must also be aware of the issues arising from false positive or negative results: the negative consequences for the patient’s health as well as the increasing number of legal claims would be unimaginable in the case of incorrect diagnosis. False positive or negative results can easily occur in case of low-grade infections and in the presence of low-virulence bacteria. Nowadays we need more specific and detailed laboratory tests on the reliability of the α-defensin assay in the diagnosis of chronic and low-grade infections, to understand the range of accuracy and therefore, it is potential role during this particular but common stage of PJI.
The interaction and collaboration between orthopedic surgeons, the medical laboratory and clinical pathologist is essential for a correct evaluation, validation and interpretation of α-defensin assay results and above all, for an accurate diagnosis of periprosthetic infections.
We should expect that some tests will move away from the central laboratory and stay closer to the patient’s bedside, but we as laboratorians should have a role to play in ensuring that the quality of results will not be diminished.
In conclusion, the management of a POCT requires dedicated resources, policies and multidisciplinary commitment and cooperation to ensure the highest quality diagnostic services.
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About the article
Published Online: 2017-07-14
Published in Print: 2017-11-27
Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
Research funding: None declared.
Employment or leadership: None declared.
Honorarium: None declared.
Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.