Tracking Anti-Staphylococcus aureus Antibodies Produced In Vivo and Ex Vivo during Foot Salvage Therapy for Diabetic Foot Infections Reveals Prognostic Insights and Evidence of Diversified Humoral Immunity.

Management of foot salvage therapy (FST) for diabetic foot infections (DFI) is challenging due to the absence of reliable diagnostics to identify the etiologic agent and prognostics to justify aggressive treatments. As Staphylococcus aureus is the most common pathogen associated with DFI, we aimed to develop a multiplex immunoassay of IgG in serum and medium enriched for newly synthesized anti-S. aureus antibodies (MENSA) generated from cultured peripheral blood mononuclear cells of DFI patients undergoing FST. Wound samples were collected from 26 DFI patients to identify the infecting bacterial species via 16S rRNA sequencing. Blood was obtained over 12 weeks of FST to assess anti-S. aureus IgG levels in sera and MENSA. The results showed that 17 out of 26 infections were polymicrobial and 12 were positive for S. aureus While antibody titers in serum and MENSA displayed similar diagnostic potentials to detect S. aureus infection, MENSA showed a 2-fold-greater signal-to-background ratio. Multivariate analyses revealed increases in predictive power of diagnosing S. aureus infections (area under the receiver operating characteristic curve [AUC] > 0.85) only when combining titers against different classes of antigens, suggesting cross-functional antigenic diversity. Anti-S. aureus IgG levels in MENSA decreased with successful FST and rose with reinfection. In contrast, IgG levels in serum remained unchanged throughout the 12-week FST. Collectively, these results demonstrate the applicability of serum and MENSA for diagnosis of S. aureus DFI with increased power by combining functionally distinct titers. We also found that tracking MENSA has prognostic potential to guide clinical decisions during FST.

Species-Specific Immunoassay Aids Identification of Pathogen and Tracks Infectivity in Foot Infection.

BACKGROUND: Conventional bacterial cultures frequently fail to identify the dominant pathogen in polymicrobial foot infections, in which Staphylococcus aureus is the most common infecting pathogen. Previous work has shown that species-specific immunoassays may be able to identify the main pathogen in musculoskeletal infections. We sought to investigate the clinical applicability of a S. aureus immunoassay to accurately identify the infecting pathogen and monitor its infectivity longitudinally in foot infection. We hypothesized that this species-specific immunoassay could aid in the diagnosis of S. aureus and track the therapeutic response in foot infections. METHODS: From July 2015 to July 2019, 83 infected foot ulcer patients undergoing surgical intervention (debridement or amputation) were recruited and blood was drawn at 0, 4, 8, and 12 weeks. Whole blood was analyzed for S. aureus-specific serum antibodies (mix of historic and new antibodies) and plasmablasts were isolated and cultured to quantify titers of newly synthesized antibodies (NSAs). Anti-S. aureus antibody titers were compared with culture results to assess their concordance in identifying S. aureus as the pathogen. The NSA titer changes at follow-ups were compared with wound healing status to evaluate concordance between evolving host immune response and clinically resolving or relapsing infection. RESULTS: Analysis of serum for anti-S. aureus antibodies showed significantly increased titers of 3 different anti-S. aureus antibodies, IsdH (P = .037), ClfB (P = .025), and SCIN (P = .005), in S. aureus culture-positive patients compared with culture-negative patients. Comparative analysis of combining antigens for S. aureus infection diagnosis increased the concordance further. During follow-up, changes of NSA titers against a single or combination of S. aureus antigens significantly correlated with clinically resolving or recurring infection represented by wound healing status. CONCLUSION: In the management of foot infection, the use of S. aureus-specific immunoassay may aid in diagnosis of the dominant pathogen and monitoring of the host immune response against a specific pathogen in response to treatment. Importantly, this immunoassay could detect recurrent foot infection, which may guide a surgeon's decision to intervene. LEVEL OF EVIDENCE: Level II, prospective comparative study.

Clinical utilization of species-specific immunoassays for identification of Staphylococcus aureus and Streptococcus agalactiae in orthopedic infections.

Staphylococcus aureus and Streptococcus agalactiae (Group B streptococcus, GBS) are common causes of deep musculoskeletal infections (MSKI) and result in significant patient morbidity and cost to the healthcare system. One of the major challenges with MSKI is the lack of faithful diagnostics to correctly identify the primary pathogen, as standard culture-based assays are prone to false positives in the case of polymicrobial infections, and false negatives due to limitations in sample acquisition and antibiotic use before presentation. To improve upon our current diagnostic methods for MSKI, we developed a multiplex immunoassay for antigen-specific IgGs in serum (Luminex), and medium enriched for newly synthesized antibodies (MENSA) for anti-S. aureus and GBS generated from cultured peripheral blood mononuclear cells (PBMCs) of orthopedic infection patients undergoing surgical treatment. Samples were obtained from 110 MSKI patients: 80 diabetic foot ulcer, 21 periprosthetic joint infection, 5 septic arthritis, 2 spine, 1 hand, and 1 fracture-related infection (FRI). Anti-S. aureus and anti-GBS antibody titers were compared to culture results to assess their concordance in identifying the pathogens. Immunoassay, particularly MENSA, showed high diagnostic potential for monomicrobial S. aureus and GBS orthopedic infections (AUC > 0.95). MENSA also demonstrated diagnostic potential for GBS polymicrobial orthopedic infection and for GBS DFU (AUC > 0.83 for both). Serum showed high diagnostic potential for S. aureus PJI (AUC > 0.95). Taken together, these findings support the development of species-specific immunoassays for the identification of causal pathogens in active MSKI, especially in conjunction with standard culture.

Calcium Phosphate Spacers for the Local Delivery of Sitafloxacin and Rifampin to Treat Orthopedic Infections: Efficacy and Proof of Concept in a Mouse Model of Single-Stage Revision of Device-Associated Osteomyelitis.

Osteomyelitis is a chronic bone infection that is often treated with adjuvant antibiotic-impregnated poly(methyl methacrylate) (PMMA) cement spacers in multi-staged revisions. However, failure rates remain substantial due to recurrence of infection, which is attributed to the poor performance of the PMMA cement as a drug release device. Hence, the objective of this study was to design and evaluate a bioresorbable calcium phosphate scaffold (CaPS) for sustained antimicrobial drug release and investigate its efficacy in a murine model of femoral implant-associated osteomyelitis. Incorporating rifampin and sitafloxacin, which are effective against bacterial phenotypes responsible for bacterial persistence, into 3D-printed CaPS coated with poly(lactic co-glycolic) acid, achieved controlled release for up to two weeks. Implantation into the murine infection model resulted in decreased bacterial colonization rates at 3- and 10-weeks post-revision for the 3D printed CaPS in comparison to gentamicin-laden PMMA. Furthermore, a significant increase in bone formation was observed for 3D printed CaPS incorporated with rifampin at 3 and 10 weeks. The results of this study demonstrate that osteoconductive 3D printed CaPS incorporated with antimicrobials demonstrate more efficacious bacterial colonization outcomes and bone growth in a single-stage revision in comparison to gentamicin-laden PMMA requiring a two-stage revision.