The 2023 Orthopaedic Research Society's International Consensus Meeting on musculoskeletal infection: Summary from the host immunity section.

Musculoskeletal infections (MSKI), which are a major problem in orthopedics, occur when the pathogen eludes or overwhelms the host immune system. While effective vaccines and immunotherapies to prevent and treat MSKI should be possible, fundamental knowledge gaps in our understanding of protective, nonprotective, and pathogenic host immunity are prohibitive. We also lack critical knowledge of how host immunity is affected by the microbiome, implants, prior infection, nutrition, antibiotics, and concomitant therapies, autoimmunity, and other comorbidities. To define our current knowledge of these critical topics, a Host Immunity Section of the 2023 Orthopaedic Research Society MSKI International Consensus Meeting (ICM) proposed 78 questions. Systematic reviews were performed on 15 of these questions, upon which recommendations with level of evidence were voted on by the 72 ICM delegates, and another 12 questions were voted on with a recommendation of "Unknown" without systematic reviews. Two questions were transferred to another ICM Section, and the other 45 were tabled for future consideration due to limitations of available human resources. Here we report the results of the voting with internet access to the questions, recommendations, and rationale from the systematic reviews. Eighteen questions received a consensus vote of ≥90%, while nine recommendations failed to achieve this threshold. Commentary on why consensus was not achieved on these questions and potential ways forward are provided to stimulate specific funding mechanisms and research on these critical MSKI host defense questions.

Circulating antibody-secreting cells are a biomarker for early diagnosis in patients with Lyme disease.

BACKGROUND: Diagnostic immunoassays for Lyme disease have several limitations including: 1) not all patients seroconvert; 2) seroconversion occurs later than symptom onset; and 3) serum antibody levels remain elevated long after resolution of the infection. INTRODUCTION: MENSA (Medium Enriched for Newly Synthesized Antibodies) is a novel diagnostic fluid that contains antibodies produced in vitro by circulating antibody-secreting cells (ASC). It enables measurement of the active humoral immune response. METHODS: In this observational, case-control study, we developed the MicroB-plex Anti-C6/Anti-pepC10 Immunoassay to measure antibodies specific for the Borrelia burgdorferi peptide antigens C6 and pepC10 and validated it using a CDC serum sample collection. Then we examined serum and MENSA samples from 36 uninfected Control subjects and 12 Newly Diagnosed Lyme Disease Patients. RESULTS: Among the CDC samples, antibodies against C6 and/or pepC10 were detected in all seropositive Lyme patients (8/8), but not in sera from seronegative patients or healthy controls (0/24). Serum antibodies against C6 and pepC10 were detected in one of 36 uninfected control subjects (1/36); none were detected in the corresponding MENSA samples (0/36). In samples from newly diagnosed patients, serum antibodies identified 8/12 patients; MENSA antibodies also detected 8/12 patients. The two measures agreed on six positive individuals and differed on four others. In combination, the serum and MENSA tests identified 10/12 early Lyme patients. Typically, serum antibodies persisted 80 days or longer while MENSA antibodies declined to baseline within 40 days of successful treatment. DISCUSSION: MENSA-based immunoassays present a promising complement to serum immunoassays for diagnosis and tracking therapeutic success in Lyme infections.

Systemic IL-27 administration prevents abscess formation and osteolysis via local neutrophil recruitment and activation.

Interleukin-27 is a pleiotropic cytokine whose functions during bacterial infections remain controversial, and its role in patients with S. aureus osteomyelitis is unknown. To address this knowledge gap, we completed a clinical study and observed elevated serum IL-27 levels (20-fold higher, P < 0.05) in patients compared with healthy controls. Remarkably, IL-27 serum levels were 60-fold higher in patients immediately following septic death than in uninfected patients (P < 0.05), suggesting a pathogenic role of IL-27. To test this hypothesis, we evaluated S. aureus osteomyelitis in WT and IL-27Rα-/- mice with and without exogenous IL-27 induction by intramuscular injection of rAAV-IL-27p28 or rAAV-GFP, respectively. We found that IL-27 was induced at the surgical site within 1 day of S. aureus infection of bone and was expressed by M0, M1 and M2 macrophages and osteoblasts but not by osteoclasts. Unexpectedly, exogenous IL-27p28 (~2 ng·mL-1 in serum) delivery ameliorated soft tissue abscesses and peri-implant bone loss during infection, accompanied by enhanced local IL-27 expression, significant accumulation of RORγt+ neutrophils at the infection site, a decrease in RANK+ cells, and compromised osteoclast formation. These effects were not observed in IL-27Rα-/- mice compared with WT mice, suggesting that IL-27 is dispensable for immunity but mediates redundant immune and bone cell functions during infection. In vitro studies and bulk RNA-seq of infected tibiae showed that IL-27 increased nos1, nos2, il17a, il17f, and rorc expression but did not directly stimulate chemotaxis. Collectively, these results identify a novel phenomenon of IL-27 expression by osteoblasts immediately following S. aureus infection of bone and suggest a protective role of systemic IL-27 in osteomyelitis.

The Serological Sciences Network (SeroNet) for COVID-19: Depth and Breadth of Serology Assays and Plans for Assay Harmonization.

In October 2020, the National Cancer Institute (NCI) Serological Sciences Network (SeroNet) was established to study the immune response to COVID-19, and "to develop, validate, improve, and implement serological testing and associated technologies" (https://www.cancer.gov/research/key-initiatives/covid-19/coronavirus-research-initiatives/serological-sciences-network). SeroNet is comprised of 25 participating research institutions partnering with the Frederick National Laboratory for Cancer Research (FNLCR) and the SeroNet Coordinating Center. Since its inception, SeroNet has supported collaborative development and sharing of COVID-19 serological assay procedures and has set forth plans for assay harmonization. To facilitate collaboration and procedure sharing, a detailed survey was sent to collate comprehensive assay details and performance metrics on COVID-19 serological assays within SeroNet. In addition, FNLCR established a protocol to calibrate SeroNet serological assays to reference standards, such as the U.S. severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serology standard reference material and first WHO international standard (IS) for anti-SARS-CoV-2 immunoglobulin (20/136), to facilitate harmonization of assay reporting units and cross-comparison of study data. SeroNet institutions reported development of a total of 27 enzyme-linked immunosorbent assay (ELISA) methods, 13 multiplex assays, and 9 neutralization assays and use of 12 different commercial serological methods. FNLCR developed a standardized protocol for SeroNet institutions to calibrate these diverse serological assays to reference standards. In conclusion, SeroNet institutions have established a diverse array of COVID-19 serological assays to study the immune response to SARS-CoV-2 and vaccines. Calibration of SeroNet serological assays to harmonize results reporting will facilitate future pooled data analyses and study cross-comparisons. IMPORTANCE SeroNet institutions have developed or implemented 61 diverse COVID-19 serological assays and are collaboratively working to harmonize these assays using reference materials to establish standardized reporting units. This will facilitate clinical interpretation of serology results and cross-comparison of research data.

The Serological Sciences Network (SeroNet) for COVID-19: Depth and Breadth of Serology Assays and Plans for Assay Harmonization.

Background: In October 2020, the National Cancer Institute (NCI) Serological Sciences Network (SeroNet) was established to study the immune response to COVID-19, and "to develop, validate, improve, and implement serological testing and associated technologies." SeroNet is comprised of 25 participating research institutions partnering with the Frederick National Laboratory for Cancer Research (FNLCR) and the SeroNet Coordinating Center. Since its inception, SeroNet has supported collaborative development and sharing of COVID-19 serological assay procedures and has set forth plans for assay harmonization. Methods: To facilitate collaboration and procedure sharing, a detailed survey was sent to collate comprehensive assay details and performance metrics on COVID-19 serological assays within SeroNet. In addition, FNLCR established a protocol to calibrate SeroNet serological assays to reference standards, such as the U.S. SARS-CoV-2 serology standard reference material and First WHO International Standard (IS) for anti-SARS-CoV-2 immunoglobulin (20/136), to facilitate harmonization of assay reporting units and cross-comparison of study data. Results: SeroNet institutions reported development of a total of 27 ELISA methods, 13 multiplex assays, 9 neutralization assays, and use of 12 different commercial serological methods. FNLCR developed a standardized protocol for SeroNet institutions to calibrate these diverse serological assays to reference standards. Conclusions: SeroNet institutions have established a diverse array of COVID-19 serological assays to study the immune response to SARS-CoV-2 virus and vaccines. Calibration of SeroNet serological assays to harmonize results reporting will facilitate future pooled data analyses and study cross-comparisons.

Detection of Newly Secreted Antibodies Predicts Nonrecurrence in Primary Clostridioides difficile Infection.

Within 8 weeks of primary Clostridioides difficile infection (CDI), as many as 30% of patients develop recurrent disease with the associated risks of multiple relapses, morbidity, and economic burden. There are no clear clinical correlates or validated biomarkers that can predict recurrence during primary infection. This study demonstrated the potential of a simple test for identifying hospitalized CDI patients at low risk for disease recurrence. Forty-six hospitalized CDI patients were enrolled at Emory University Hospitals. Samples of serum and a novel matrix from circulating plasmablasts called "medium-enriched for newly synthesized antibodies" (MENSA) were collected during weeks 1, 2, and 4. Antibodies specific for 10 C. difficile antigens were measured in each sample. Among the 46 C. difficile-infected patients, 9 (19.5%) experienced recurrence within 8 weeks of primary infection. Among the 37 nonrecurrent patients, 23 (62%; 23/37) had anti-C. difficile MENSA antibodies specific for any of the three toxin antigens: TcdB-CROP, TcdBvir-CROP, and/or CDTb. Positive MENSA responses occurred early (within the first 12 days post-symptom onset), including six patients who never seroconverted. A similar trend was observed in serum responses, but they peaked later and identified fewer patients (51%; 19/37). In contrast, none (0%; 0/9) of the patients who subsequently recurred after hospitalization produced antibodies specific for any of the three C. difficile toxin antigens. Thus, patients with a negative early MENSA response against all three C. difficile toxin antigens had a 19-fold greater relative risk of recurrence. MENSA and serum levels of immunoglobulin A (IgA) and/or IgG antibodies for three C. difficile toxins have prognostic potential. These immunoassays measure nascent immune responses that reduce the likelihood of recurrence thereby providing a biomarker of protection from recurrent CDI. Patients who are positive by this immunoassay are unlikely to suffer a recurrence. Early identification of patients at risk for recurrence by negative MENSA creates opportunities for targeted prophylactic strategies that can reduce the incidence, cost, and morbidity due to recurrent CDI.

Diagnosis of Streptococcus pneumoniae infection using circulating antibody secreting cells.

BACKGROUND: Streptococcus pneumoniae infections cause morbidity and mortality worldwide. A rapid, simple diagnostic method could reduce the time needed to introduce definitive therapy potentially improving patient outcomes. METHODS: We introduce two new methods for diagnosing S. pneumoniae infections by measuring the presence of newly activated, pathogen-specific, circulating Antibody Secreting Cells (ASC). First, ASC were detected by ELISpot assays that measure cells secreting antibodies specific for signature antigens. Second, the antibodies secreted by isolated ASC were collected in vitro in a novel matrix, MENSA (media enriched with newly synthesized antibodies) and antibodies against S. pneumoniae antigens were measured using Luminex immunoassays. Each assay was evaluated using blood from S. pneumoniae and non-S. pneumoniae-infected adult patients. RESULTS: We enrolled 23 patients with culture-confirmed S. pneumoniae infections and 24 controls consisting of 12 non-S. pneumoniae infections, 10 healthy donors and two colonized with S. pneumoniae. By ELISpot assays, twenty-one of 23 infected patients were positive, and all 24 controls were negative. Using MENSA samples, four of five S. pneumoniae-infected patients were positive by Luminex immunoassays while all five non-S. pneumoniae-infected patients were negative. CONCLUSION: Specific antibodies produced by activated ASC may provide a simple diagnostic for ongoing S. pneumoniae infections. This method has the potential to diagnose acute bacterial infections.

StaphAIR: A Label-Free Antigen Microarray Approach to Detecting Anti-Staphylococcus aureus Antibody Responses in Orthopedic Infections.

Arrayed imaging reflectometry (AIR) is an optical biosensor platform for simple, multiplex measurement of antigen-specific antibody responses in patient blood samples. Here, we report the development of StaphAIR, an 8-plex Staphylococcus aureus antigen array on the AIR platform for profiling antigen-specific anti-S. aureus humoral immune responses. Initial validation experiments with mouse and humanized monoclonal antibodies against the S. aureus autolysin glucosaminidase (Gmd) domain, and subsequent testing with dilution series of pooled positive human serum confirmed analytically robust behavior of the array, with all antigens displaying Langmuir-type dose-response curves. Testing a cohort of 82 patients with S. aureus musculoskeletal infections (MSKI) and 30 healthy individuals enabled discrimination of individual patient responses to different S. aureus antigens, with statistical significance between osteomyelitis patients and controls obtained overall for four individual antigens (IsdA, IsdB, Gmd, and SCIN). Multivariate analyses of the antibody titers obtained from StaphAIR revealed its utility as a potential diagnostic tool for detecting S. aureus MSKI (area under the receiver operating characteristic curve (AUC) > 0.85). We conclude that StaphAIR has utility as a high-throughput immunoassay for studying and diagnosing osteomyelitis in patients.

One-Stop Serum Assay Identifies COVID-19 Disease Severity and Vaccination Responses.

SARS-CoV-2 has caused over 100,000,000 cases and almost 2,500,000 deaths globally. Comprehensive assessment of the multifaceted antiviral Ab response is critical for diagnosis, differentiation of severity, and characterization of long-term immunity, especially as COVID-19 vaccines become available. Severe disease is associated with early, massive plasmablast responses. We developed a multiplex immunoassay from serum/plasma of acutely infected and convalescent COVID-19 patients and prepandemic and postpandemic healthy adults. We measured IgA, IgG, and/or IgM against SARS-CoV-2 nucleocapsid (N), spike domain 1 (S1), S1-receptor binding domain (RBD) and S1-N-terminal domain. For diagnosis, the combined [IgA + IgG + IgM] or IgG levels measured for N, S1, and S1-RBD yielded area under the curve values ≥0.90. Virus-specific Ig levels were higher in patients with severe/critical compared with mild/moderate infections. A strong prozone effect was observed in sera from severe/critical patients-a possible source of underestimated Ab concentrations in previous studies. Mild/moderate patients displayed a slower rise and lower peak in anti-N and anti-S1 IgG levels compared with severe/critical patients, but anti-RBD IgG and neutralization responses reached similar levels at 2-4 mo after symptom onset. Measurement of the Ab responses in sera from 18 COVID-19-vaccinated patients revealed specific responses for the S1-RBD Ag and none against the N protein. This highly sensitive, SARS-CoV-2-specific, multiplex immunoassay measures the magnitude, complexity, and kinetics of the Ab response and can distinguish serum Ab responses from natural SARS-CoV-2 infections (mild or severe) and mRNA COVID-19 vaccines.

Humanized Mice Exhibit Exacerbated Abscess Formation and Osteolysis During the Establishment of Implant-Associated Staphylococcus aureus Osteomyelitis.

Staphylococcus aureus is the predominant pathogen causing osteomyelitis. Unfortunately, no immunotherapy exists to treat these very challenging and costly infections despite decades of research, and numerous vaccine failures in clinical trials. This lack of success can partially be attributed to an overreliance on murine models where the immune correlates of protection often diverge from that of humans. Moreover, S. aureus secretes numerous immunotoxins with unique tropism to human leukocytes, which compromises the targeting of immune cells in murine models. To study the response of human immune cells during chronic S. aureus bone infections, we engrafted non-obese diabetic (NOD)-scid IL2Rγnull (NSG) mice with human hematopoietic stem cells (huNSG) and analyzed protection in an established model of implant-associated osteomyelitis. The results showed that huNSG mice have increases in weight loss, osteolysis, bacterial dissemination to internal organs, and numbers of Staphylococcal abscess communities (SACs), during the establishment of implant-associated MRSA osteomyelitis compared to NSG controls (p < 0.05). Flow cytometry and immunohistochemistry demonstrated greater human T cell numbers in infected versus uninfected huNSG mice (p < 0.05), and that T-bet+ human T cells clustered around the SACs, suggesting S. aureus-mediated activation and proliferation of human T cells in the infected bone. Collectively, these proof-of-concept studies underscore the utility of huNSG mice for studying an aggressive form of S. aureus osteomyelitis, which is more akin to that seen in humans. We have also established an experimental system to investigate the contribution of specific human T cells in controlling S. aureus infection and dissemination.

Novel immunoassay for diagnosis of ongoing Clostridioides difficile infections using serum and medium enriched for newly synthesized antibodies (MENSA).

BACKGROUND: Clostridioides difficile infections (CDI) have been a challenging and increasingly serious concern in recent years. While early and accurate diagnosis is crucial, available assays have frustrating limitations. OBJECTIVE: Develop a simple, blood-based immunoassay to accurately diagnose patients suffering from active CDI. MATERIALS AND METHODS: Uninfected controls (N = 95) and CDI patients (N = 167) were recruited from Atlanta area hospitals. Blood samples were collected from patients within twelve days of a positive CDI test and processed to yield serum and PBMCs cultured to yield medium enriched for newly synthesized antibodies (MENSA). Multiplex immunoassays measured Ig responses to ten recombinant C. difficile antigens. RESULTS: Sixty-six percent of CDI patients produced measurable responses to C. difficile antigens in their serum or MENSA within twelve days of a positive CDI test. Fifty-two of the 167 CDI patients (31%) were detectable in both serum and MENSA, but 32/167 (19%) were detectable only in MENSA, and 27/167 (16%) were detectable only in serum. DISCUSSION: We describe the results of a multiplex immunoassay for the diagnosis of ongoing CDI in hospitalized patients. Our assay resolved patients into four categories: MENSA-positive only, serum-positive only, MENSA- and serum-positive, and MENSA- and serum-negative. The 30% of patients who were MENSA-positive only may be accounted for by nascent antibody secretion prior to seroconversion. Conversely, the serum-positive only subset may have been more advanced in their disease course. Immunocompromise and misdiagnosis may have contributed to the 34% of CDI patients who were not identified using MENSA or serum immunoassays. IMPORTANCE: While there was considerable overlap between patients identified through MENSA and serum, each method detected a distinctive patient group. The combined use of both MENSA and serum to detect CDI patients resulted in the greatest identification of CDI patients. Together, longitudinal analysis of MENSA and serum will provide a more accurate evaluation of successful host humoral immune responses in CDI patients.

Bacterial toxins in musculoskeletal infections.

Musculoskeletal infections (MSKIs) remain a major health burden in orthopaedics. Bacterial toxins are foundational to pathogenesis in MSKI, but poorly understood by the community of providers that care for patients with MSKI, inducing an international group of microbiologists, infectious diseases specialists, orthopaedic surgeons and biofilm scientists to review the literature in this field to identify key topics and compile the current knowledge on the role of toxins in MSKI, with the goal of illuminating potential impact on biofilm formation and dispersal as well as therapeutic strategies. The group concluded that further research is needed to maximize our understanding of the effect of toxins on MSKIs, including: (i) further research to identify the roles of bacterial toxins in MSKIs, (ii) establish the understanding of the importance of environmental and host factors and in vivo expression of toxins throughout the course of an infection, (iii) establish the principles of drug-ability of antitoxins as antimicrobial agents in MSKIs, (iv) have well-defined metrics of success for antitoxins as antiinfective drugs, (v) design a cocktail of antitoxins against specific pathogens to (a) inhibit biofilm formation and (b) inhibit toxin release. The applicability of antitoxins as potential antimicrobials in the era of rising antibiotic resistance could meet the needs of day-to-day clinicians.

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.

Serum antibodies against Staphylococcus aureus can prognose treatment success in patients with bone infections.

Prognosing life-threatening orthopedic infections caused by Staphylococcus aureus remains a major clinical challenge. To address this, we developed a multiplex assay to assess the humoral immune proteome against S. aureus in patients with musculoskeletal infections. We found initial evidence that antibodies against some antigens (autolysins: Amd, Gmd; secreted immunotoxins: CHIPS, SCIN, Hla) were associated with protection, whereas antibodies against the iron-regulated surface determinant (Isd) proteins (IsdA, IsdB, IsdH) were aligned with adverse outcomes. To formally test this, we analyzed antibody levels and 1-year clinical outcomes of 194 patients with confirmed S. aureus bone infections (AO Trauma Clinical Priority Program [CPP] Bone Infection Registry). A staggering 20.6% of the enrolled patients experienced adverse clinical outcomes (arthrodesis, reinfection, amputation, and septic death) after 1-year. At enrollment, anti-S. aureus immunoglobulin G (IgG) levels in patients with adverse outcomes were 1.35-fold lower than those in patients whose infections were successfully controlled (p < 0.0001). Overall, there was a 51%-69% reduction in adverse outcome risk for every 10-fold increase in initial IgG concentration against Gmd, Amd, IsdH, CHIPS, SCIN, and Hla (p < 0.05). Notably, anti-IsdB antibodies remained elevated in patients with adverse outcomes; for every 10-fold change in the ratio of circulating anti-Isd to anti-Atl IgG at enrollment, there was a trending 2.6-fold increased risk (odds ratio = 2.555) of an adverse event (p = 0.105). Moreover, antibody increases over time correlated with adverse outcomes and decreases with positive outcomes. These studies demonstrate the potential of the humoral immune response against S. aureus as a prognostic indicator for assessing treatment success and identifying patients requiring additional interventions.

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.

Distinct expression trend of signature antigens of Staphylococcus aureus osteomyelitis correlated with clinical outcomes.

The major limitations of clinical outcome predictions of osteomyelitis mediated by Staphylococcus aureus (S. aureus) are not specific and definitive. To this end, current studies aim to investigate host immune responses of trend changes of the iron-regulated surface determinant (Isd) of IsdA, IsdB, IsdH, cell wall-modifying proteins of amidase (Amd) and glucosaminidase (Gmd), and secreted virulence factor of chemotaxis inhibitory protein S. aureus (CHIPS) and staphylococcal complement inhibitor (SCIN) longitudinally to discover their correlationship with clinical outcomes. A total of 55 patients with confirmed S. aureus infection of the long bone by clinical and laboratory methods were recruited for the study. Whole blood was collected at 0, 6, 12 months for the serum that was used to test IsdA, IsdB, IsdH, Gmd, Amd, CHIPS, and SCIN using a customized Luminex assay after clinical standard care parameters were collected. The patients were then divided into two groups: (1) infection controlled versus (2) adverse outcome based on clinical criteria for statistical analysis. We found that standard clinical parameters were unable to distinguish therapeutic outcomes. Significant overexpression of all antigens was confirmed in infection patients at 0-, 6-, and 12-month time points. A distinct expression trend and dynamic changes of IsdB, Amd, Gmd, and CHIPS were observed between infection controlled and adverse outcome patients, while the IsdA, IsdH, SCIN remained demonstrated no statistical significance. We conclude that dynamic changes of specific antigens could predict clinical outcomes of S. aureus osteomyelitis. Clinical Relevance: The trend changes of host immune responses to S. aureus specific antigens of IsdB, Gmd, Amd, and CHIPS could predict clinical outcomes of S. aureus osteomyelitis.

Elevated SARS-CoV-2 Antibodies Distinguish Severe Disease in Early COVID-19 Infection.

BACKGROUND: SARS-CoV-2 has caused over 36,000,000 cases and 1,000,000 deaths globally. Comprehensive assessment of the multifaceted anti-viral antibody response is critical for diagnosis, differentiation of severe disease, and characterization of long-term immunity. Initial observations suggest that severe disease is associated with higher antibody levels and greater B cell/plasmablast responses. A multi-antigen immunoassay to define the complex serological landscape and clinical associations is essential. METHODS: We developed a multiplex immunoassay and evaluated serum/plasma from adults with RT-PCR-confirmed SARS-CoV-2 infections during acute illness (N=52) and convalescence (N=69); and pre-pandemic (N=106) and post-pandemic (N=137) healthy adults. We measured IgA, IgG, and/or IgM against SARS-CoV-2 Nucleocapsid (N), Spike domain 1 (S1), receptor binding domain (S1-RBD) and S1-N-terminal domain (S1-NTD). RESULTS: To diagnose infection, the combined [IgA+IgG+IgM] or IgG for N, S1, and S1-RBD yielded AUC values -0.90 by ROC curves. From days 6-30 post-symptom onset, the levels of antigen-specific IgG, IgA or [IgA+IgG+IgM] were higher in patients with severe/critical compared to mild/moderate infections. Consistent with excessive concentrations of antibodies, a strong prozone effect was observed in sera from severe/critical patients. Notably, mild/moderate patients displayed a slower rise and lower peak in anti-N and anti-S1 IgG levels compared to severe/critical patients, but anti-RBD IgG and neutralization responses reached similar levels at 2-4 months. CONCLUSION: This SARS-CoV-2 multiplex immunoassay measures the magnitude, complexity and kinetics of the antibody response against multiple viral antigens. The IgG and combined-isotype SARS-CoV-2 multiplex assay is highly diagnostic of acute and convalescent disease and may prognosticate severity early in illness. ONE SENTENCE SUMMARY: In contrast to patients with moderate infections, those with severe COVID-19 develop prominent, early antibody responses to S1 and N proteins.

IsdB antibody-mediated sepsis following S. aureus surgical site infection.

Staphylococcus aureus is prevalent in surgical site infections (SSI) and leads to death in approximately 1% of patients. Phase IIB/III clinical trial results have demonstrated that vaccination against the iron-regulated surface determinant protein B (IsdB) is associated with an increased mortality rate in patients with SSI. Thus, we hypothesized that S. aureus induces nonneutralizing anti-IsdB antibodies, which facilitate bacterial entry into leukocytes to generate "Trojan horse" leukocytes that disseminate the pathogen. Since hemoglobin (Hb) is the primary target of IsdB, and abundant Hb-haptoglobin (Hb-Hp) complexes in bleeding surgical wounds are normally cleared via CD163-mediated endocytosis by macrophages, we investigated this mechanism in vitro and in vivo. Our results demonstrate that active and passive IsdB immunization of mice renders them susceptible to sepsis following SSI. We also found that a multimolecular complex containing S. aureus protein A-anti-IsdB-IsdB-Hb-Hp mediates CD163-dependent bacterial internalization of macrophages in vitro. Moreover, IsdB-immunized CD163-/- mice are resistant to sepsis following S. aureus SSI, as are normal healthy mice given anti-CD163-neutralizing antibodies. These genetic and biologic CD163 deficiencies did not exacerbate local infection. Thus, anti-IsdB antibodies are a risk factor for S. aureus sepsis following SSI, and disruption of the multimolecular complex and/or CD163 blockade may intervene.

Extrafollicular B cell responses correlate with neutralizing antibodies and morbidity in COVID-19.

A wide spectrum of clinical manifestations has become a hallmark of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) COVID-19 pandemic, although the immunological underpinnings of diverse disease outcomes remain to be defined. We performed detailed characterization of B cell responses through high-dimensional flow cytometry to reveal substantial heterogeneity in both effector and immature populations. More notably, critically ill patients displayed hallmarks of extrafollicular B cell activation and shared B cell repertoire features previously described in autoimmune settings. Extrafollicular activation correlated strongly with large antibody-secreting cell expansion and early production of high concentrations of SARS-CoV-2-specific neutralizing antibodies. Yet, these patients had severe disease with elevated inflammatory biomarkers, multiorgan failure and death. Overall, these findings strongly suggest a pathogenic role for immune activation in subsets of patients with COVID-19. Our study provides further evidence that targeted immunomodulatory therapy may be beneficial in specific patient subpopulations and can be informed by careful immune profiling.

Lack of Humoral Immunity Against Glucosaminidase Is Associated with Postoperative Complications in Staphylococcus aureus Osteomyelitis.

BACKGROUND: Glucosaminidase (Gmd) is known to be a protective antigen in animal models of Staphylococcus aureus osteomyelitis. We compared the endogenous anti-Gmd antibody levels in sera of patients with culture-confirmed S. aureus bone infections to their sera at 1 year after operative treatment of the infection. METHODS: A novel global biospecimen registry of 297 patients with deep-wound culture-confirmed S. aureus osteomyelitis was analyzed to assess relationships between baseline anti-Gmd serum titers (via custom Luminex assay), known host risk factors for infection, and 1-year postoperative clinical outcomes (e.g., infection control, inconclusive, refracture, persistent infection, septic nonunion, amputation, and septic death). RESULTS: All patients had measurable humoral immunity against some S. aureus antigens, but only 20 patients (6.7%; p < 0.0001) had high levels of anti-Gmd antibodies (>10 ng/mL) in serum at baseline. A subset of 194 patients (65.3%) who completed 1 year of follow-up was divided into groups based on anti-Gmd level: low (<1 ng/mL, 54 patients; 27.8%), intermediate (<10 ng/mL, 122 patients; 62.9%), and high (>10 ng/mL, 18 patients; 9.3%), and infection control rates were 40.7%, 50.0%, and 66.7%, respectively. The incidence of adverse outcomes in these groups was 33.3%, 16.4%, and 11.1%, respectively. Assessing anti-Gmd level as a continuous variable showed a 60% reduction in adverse-event odds (p = 0.04) for every tenfold increase in concentration. No differences in patient demographics, body mass index of >40 kg/m, diabetes status, age of ≥70 years, male sex, Charlson Comorbidity Index of >1, or Cierny-Mader host type were observed between groups, and these risk factors were not associated with adverse events. Patients with low anti-Gmd titer demonstrated a significant 2.68-fold increased odds of adverse outcomes (p = 0.008). CONCLUSIONS: Deficiency in circulating anti-Gmd antibodies was associated serious adverse outcomes following operative treatment of S. aureus osteomyelitis. At 1 year, high levels of anti-Gmd antibodies were associated with a nearly 3-fold increase in infection-control odds. Additional prospective studies clarifying Gmd immunization for osteomyelitis are needed. LEVEL OF EVIDENCE: Prognostic Level IV. See Instructions for Authors for a complete description of levels of evidence.