Anti-glucosaminidase IgG in sera as a biomarker of host immunity against Staphylococcus aureus in orthopaedic surgery patients.

Postoperative infection remains a major complication of orthopaedic surgery. Although infections are caused by an array of microorganisms, most involve Staphylococcus aureus1,2. In total joint replacement, up to 80% of infections involve staphylococci, which can be divided into coagulase-positive and coagulase-negative species3. Most non-staphylococci and many coagulase-negative staphylococci respond well to antibiotic therapy. In contrast, infections by Staphylococcus aureus (especially methicillin-resistant Staphylococcus aureus [MRSA]) are resistant to antibiotics and represent the most challenging and costly clinical cases4-6. Timely and accurate diagnosis of Staphylococcus aureus infections is challenging3,7,8. Although the American Academy of Orthopaedic Surgeons has recently established guidelines for the diagnosis of periprosthetic infection on the basis of the blood ESR (erythrocyte sedimentation rate) and CRP (C-reactive protein) level combined with frozen sections and joint aspiration9, diagnosis remains difficult for patients who are culture-negative as a result of antibiotic therapy but may have a deep biofilm-mediated infection undetectable with use of tissue samples10. There is no generally accepted confirmatory test for infection. Available methods include serum biomarkers such as CRP, interleukin-6, and procalcitonin11; ESR12; blood and joint fluid leukocyte counts; culture and PCR (polymerase chain reaction) tests for the pathogens3,13; and radiographic imaging techniques that measure bone lysis or scintigraphic measurements of localized neutrophil accumulation14,15. None of these tests has the combination of simplicity, cost-effectiveness, and predictive power necessary to make it routinely applicable, especially for the diagnosis of early-stage infections. Furthermore, although host immunity is predictive of a patient’s susceptibility to serious infection or clinical outcome of osteomyelitis, there is no diagnostic test for host immunity against Staphylococcus aureus. A diagnostic approach for Staphylococcus aureus infection that has not been explored is the evaluation of serum immunoglobulin G (IgG) similar to that performed routinely for viral infections such as HIV (human immunodeficiency virus). The “immune proteome” hypothesis, based on IgG responses in animal models and patients, has emerged as an explanation of how mammals protect themselves from Staphylococcus aureus infection16,17. This theory posits that (1) Staphylococcus aureus proteins can be grouped into distinct functional categories such as secreted toxins (α-hemolysin, Panton-Valentine leukocidin), iron-scavenging proteins (IsdA, IsdB, IsdH), and cell-wall adhesins (ClfA, FnbpA); and (2) effective humoral immunity requires the generation of a host antibody response against a constellation of these antigens. Our research has focused on the cell-wall-modifying enzyme AtlA, and it is based on our identification of the glucosaminidase (Gmd) domain of AltA as a potentially protective antigen in a murine model of implant-associated osteomyelitis18. We subsequently generated recombinant histidine-tagged Gmd protein (His-Gmd) and used it as a vaccine to generate neutralizing anti-Gmd IgG1 monoclonal antibodies (mAb 1C11 and 4A12)19. With the goal of developing a rapid serum diagnostic test to detect Staphylococcus aureus infection on the basis of antigen-specific IgG titers, we utilized these monoclonal antibodies to validate in vitro assays that quantify physical titers (measuring IgG binding to His-Gmd) and functional titers (measuring the ability to inhibit the enzymatic activity of His-Gmd (cell-wall digestion). These novel assays were then used to test the hypothesis that anti-Gmd IgG titers would be diagnostic of Staphylococcus aureus infection in a small cohort of orthopaedic patients.