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

Abstract

BACKGROUND

Conventional culture frequently fails 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 can 1) aid diagnosis of S. aureus and 2) 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 (NSA). Anti-S. aureus antibody titers were compared to culture results to assess their concordance in identifying S. aureus as the pathogen. The NSA titer changes at follow-ups were compared to wound healing status to evaluate concordance between evolving host immune response versus 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 = 0.037), ClfB (p = 0.025), and SCIN (p = 0.005) in S. aureus culture positive patients compared to culture negative patients. Comparative analysis of combining antigens for S. aureus infection diagnosis increased the concordance further. During follow-ups, 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 statuses. In summary, this S. aureus immunoassay was able to track treatment response and detect recurrent infection.

CONCLUSIONS

In management of foot infection, the use of S. aureus-specific immunoassay may aid in diagnosis of the dominant pathogen and monitor the host immune response against a specific pathogen in response to treatment. Importantly, this immunoassay can detect recurrent foot infection which may guide a surgeon’s decision to intervene.

Level II:

Diagnostic Study

INTRODUCTION

Background

Infected diabetic foot ulcer (DFU) is one of the most common disorders necessitating hospital admission and its incidence continues to rise.² Two-thirds of lower extremity amputations are associated with infected DFU.^{15, 22, 28} Most patients with diabetic foot infections present with poor glycemic control and multiple comorbidities. Additionally, these patients exhibit high rates of post-operative complications, such as surgical wound dehiscence (50%) and recurrent ulcer (40-43%), which often lead to revision surgeries (40%), including amputation.^1,2,5,21,29 DFU often present as complex polymicrobial infections,^7,8,27 where Staphylococcus aureus is the most common offending agent (46-68%), followed by coagulase-negative Staphylococci, Streptococcus agalactiae, Staphylococcus epidermidis, and Enterococcus.^{7–9,12,16,17,20,22,23,25} The conventional diagnostic microbial culture is subject to sampling error, which may yield a false-positive or false-negative growth, especially if the patient has been on antibiotics prior to obtaining sample.^{2,6,11,17,35–37} Moreover, culture cannot always differentiate dominant pathogens from benign commensals or bystanders, which may complicate antibiotic treatment after surgery.^12,35–37 Previously studies investigating prognostic markers for monitoring infection of DFUs have been inconsistent.^3,4,38,39 Most importantly, inflammatory markers such as erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) are not pathogen specific.^20,34 Marmor et al. previously reported successful utilization of a commercially available serum based species-specific immunoassay for identification of dominant pathogens in prosthetic joint infections.¹⁹ Yet, currently, there are no sensitive and specific tools for diagnosing and monitoring a pathogen’s ongoing infectivity.^24,31

Given the above challenges, we sought to investigate the applicability of a species-specific immunoassay to supplement standard culture in identification of dominant pathogens and tracking therapeutic response. Specifically, we compared titers of serum antibodies versus newly synthesized antibodies (NSA) produced by stimulated plasmabasts or antibody secreting cells (ASC) that emerge into blood only during active infection.^{9,13,14,20,22,24} Serum antibody titers include all circulating, pre-existing antibodies potentially made in response to historical exposure to pathogens, whereas NSA titers represent antibodies that are newly generated in response to a recent infection (< 2-3 weeks). Therefore, NSA immunoassay may able to accurately differentiate the dominant pathogen perceived by the host’s immune system among various polymicrobial organisms identified by a tissue culture.²⁴ Given that our immunologic response is not site-specific, we aimed to utilize this information to supplement bone culture obtained from an infected foot. In our pilot study of 26 patients with infected DFU, we found 70% concordance rate between the S. aureus immunoassay and standard culture in identification of S. aureus infection.²⁴ Moreover, the NSA immunoassay showed promise for monitoring treatment response and detecting recurrent S. aureus infection.²⁴ Most notably, in some patients who were on a 6-week course of postoperative antibiotics treatment, the 8-week follow-up (2 weeks after cessation of antibiotics treatment) immunoassay revealed persistently elevated or resurging immune response against S. aureus, which concurred with clinical recurrent infection that necessitated unexpected readmission and reoperation.²⁴ We noted that the immunoassay was more sensitive and specific than traditional inflammatory markers (ESR, CRP) in reflecting recurrent infection at an earlier phase of recurrent infection.²⁴ Given the high rate of infection recurrence in DFU and often ambiguous clinical presentation of indolent or recurrent infection, accurate assessment of evolving pathogenicity may improve DFU patients management. NSA immunoassay may provide surgeons with additional information to accurately assess treatment progression and prospect for a limb salvage.

In this study, we expand on previous work and investigate the clinical applicability of species-specific immunoassay to supplement culture in identification of S. aureus infection and monitor its infectivity in response to surgical treatment. We hypothesized that this species-specific immunoassay can 1) aid in diagnosis of S. aureus and 2) track treatment response in foot infection.

PATIENTS/METHODS

Patient Enrollment

From July 2015 to July 2019, we enrolled 83 patients who displayed clinical symptoms and signs of infected DFU^7,8 which necessitated hospitalization and surgical interventions, such as irrigation and debridement or amputation, followed by a course of IV or oral antibiotic treatment. Patients presenting with severe ischemia (ankle-brachial index < 0.45), venous stasis ulcers, pregnancy, malnutrition (albumin < 3g/dl), immune deficiency, or inability to make decision (unconscious, nonconsentable) were excluded from the study. Demographic information is summarized in Table 1.

Table 1:

Patients demographic and surgical procedures performed.

Characteristic	Values
Male:Female	60 (72.3%): 23 (27.7%)
Age (Yrs)	56.5 ± 11.7
BMI (kg/m2)	35 ± 7.4
WBC (x 103 /μl)	11.439 ± 4.441
ESR (mm/hr)	61 ± 38.8
CRP (mg/L)	112 ± 105
A1c Levels	9.3 ± 2.8
Wound Healing (% healed)	53%
Surgeries Performed:
Irrigation & Debridement (%)	25 (30%)
Toe Amputation	16 (19%)
Metatarsal/Ray Amputation	22 (27%)
Midfoot Amputation	3 (4%)
Calcanectomy	10 (12%)
Syme Amputation	1 (1%)
Below the Knee Amputation	6 (7%)

Operative Procedures and Postoperative Antibiotics Therapy

When possible, infected DFUs were initially managed by wound debridement followed by daily wet-to-dry dressing change with normal saline solution. For those who presented with extensive soft tissue and bone necrosis, amputation of the infected part was necessary. Biomechanical derangement such as equinus or isolated gastrocnemius contracture was addressed by performing concomitant achilles tendon lengthening or gastrocnemius recession as indicated. In all cases, bone samples were obtained from the base of the infected foot ulcer at the time of surgery. Pathologic evaluation of the bone resection margin was performed for all patients who underwent amputation or bony resection (i. e. calcanectomy) procedure. If clean margins were obtained, patients were placed on oral antibiotics for an additional 2 weeks to eradicate residual soft tissue infection. If the resection margins were contaminated, a targeted long-term (6 weeks) IV or oral antibiotics were instituted as recommended by the consulting infectious disease specialist. The end point of the study for each subject was at the 12-weeks post-surgical intervention or at the time of reoperation as a consequence of treatment failure, whichever occurred first. Healing wound was defined as completely healed or decreased size and depth of the wound and absence of 12 secondary signs and symptoms of infection (pain, erythema, edema, heat, purulent exudate, serous exudate with concurrent inflammation, delayed healing, dislocation of granulation tissue, friable granulation tissue, pocketing at the baes of the wound, foul odor and wound breakdown) as previously described.^7,8,24 A non-healing wound is defined as 1) unimproved or 2) increased in size and depth of the wound with persistent 12 signs and symptoms of infection.^7,8,24 During the postoperative period, various biomechanical offloading strategies such as total contact cast, pneumatic boot, plastazote insoles or soft cushioned post op shoe were utilized to offload the operated foot as deemed appropriate by the treating surgeon.

Whole Blood Processing

Three tubes (2 green-topped sodium heparin tubes and 1 red-topped serum tube) of whole blood were drawn from the study subjects at the recruitment (t = 0), 4-, 8-, and 12 weeks afterwards. The green-topped tubes were immediately processed to isolate peripheral blood mononuclear cells (PBMCs). The isolated PBMCs were subsequently cultured to populate recently activated circulating plasmablasts (or ASC) specific for S. aureus.^14,20,22,24 Serum, which contains mix of pre-existing and newly made antibodies, was collected from the red-topped tube following centrifugation. The secreted products (NSA) from cultured plasmablasts and the collected sera were stored at −80 °C until immunoassay (Figure 1A). As part of standard clinical care, blood serum samples were collected for ESR, CRP, White Blood Counts (WBC), and non-fasting glucose levels (Table 1).

Figure 1.

NSA versus Serum Immunoassay. (A) Illustration of newly synthesized antibody (NSA) medium and serum preparations for immunoassay. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood (green top) and cultured to propagate recently activated plasmablasts or antibody secreting cells, which generates newly-synthesized antibodies. Serum is isolated from whole blood (red top) to harvest mix of pre-existing and newly-made antibodies. (B) Illustration of multiplex Luminex immunoassay. Avidin-coated magnetic bead is coupled to a recombinant S. aureus antigen. We utilized 8 different beads with unique spectral signatures to couple with 8 different S. aureus specific antigens. After incubating with serum or NSA medium, human antibody against a S. aureus specific antigen forms a complex. Subsequently, the secondary phycoerythrin-conjugated goat anti-human IgG binds to the complex and run on flow cytometer to measure fluorescence intensity of the beads (to discern different beads couple with unique antigens) and phycoerythrin for quantitative measure.

Multiplex Luminex Immunoassay

Anti-S. aureus antibodies levels in the DFU patients were determined using a custom multiplex immunoassay.²⁴ Briefly, avidin-coated magnetic LumAvidin™ microspheres (Luminex, Austin, TX) with unique spectral signatures were coupled to individual recombinant S. aureus antigen.²⁴ S. aureus antigens included 1) Iron acquisition proteins – iron-regulated surface determinant proteins (IsdB, IsdH), 2) Cell division proteins – glucosaminidase (Gmd), amidase (Amd), 3) Staphylococcal inhibitory protein (SCIN), 4) Cell attachment proteins – clumping factor B (ClfB), and Panton-Valentine leukocidin subunits (LukS-PV, LukF-PV).²⁴ For detecting species-specific antigen-reactive IgG, 1,000 magnetic microspheres per analyte per well were mixed and incubated with 1) 100 μl of diluted serum (1:10,000) or 2) 100 μl of NSA medium for 2 hours. After washing, the secondary phycoerythrin-conjugated goat anti-human IgG (Southern Biotech, Birmingham, AL) was added and incubated for 1 hour. All serum and NSA samples were run on a flow cytometer (Bio-Plex 200; Bio-Rad, Hercules, CA). The fluorescence intensity of the beads and phycoerythrin (100 beads per analyte per well) were acquired for analysis (Figure 1B).

Statistical Data Analysis

S. aureus specific antibodies measurements obtained from serum and NSA medium at the recruitment (0 week) were compared to intra-operatively obtained bone culture for identification of S. aureus. The concordance of the two was assessed using receiver operating characteristic (ROC) curve analysis, with overall accuracy summarized by the area under the ROC curve (AUC). The subsequent changes of NSA titers at follow-up visits (4-, 8-, and 12 weeks) were comparatively analyzed to the clinical wound healing status. We expected to observe decreasing NSA against S. aureus (less stimulation of ASC with decreased pathogenicity) associated with clinically subsiding infection represented by healing foot ulcer or surgical incision. In contrast, persistently elevated or rebounding NSA against S. aureus antigens was expected in persistent or recurrent infection represented by nonhealing ulcer. Relative changes of baseline to follow-up antibody titers were analyzed against clinical healing versus non-healing wound status. ROC curves were analyzed for each individual and combinations of antibodies against 8 different S. aureus antigens. Combinations were formed using best subsets selection with multivariate logistic regression models to identify groups of antibodies that best diagnosed and tracked infection and healing response. The estimated linear predictor from each of these logistic models combined multiple antibodies into a single numerical score for analysis. Nonparametric estimates and 95% confidence intervals for the AUC were computed for each predictor. P-values for testing the significance of each AUC, and for comparing AUCs across predictors, were also calculated. Analyses were conducted using SAS version 9.4, and R version 3.5.1. A p-value less than 0.05 was considered significant.

RESULTS

Patient Demographics

Eighty-three infected DFU patients formed the basis of this study (Table 1). There were 23 female and 60 male participants with mean age of 56.5 (range, 26 to 82) years-old. Twenty five patients (30%) underwent wound debridement and 58 patients (70%) underwent amputations of the infected part (16 toe amputations (19%), 22 metatarsal amputation (27%), 3 midfoot amputation (4%), 10 calcanectomy (12%), 1 Syme amputation (1%) and 6 below the knee amputation (BKA) (7%)). Forty-five participants (54%) grew S. aureus in their standard culture (SA+) whereas 38 (46%) patients did not grow S. aureus (SA−). Four participants passed away prior to their final 12-week follow up due to other medical complications. At the final follow-up, 56% of SA+ participants and 46% of SA− participants healed the DFU or surgical incision without sign of recurrent infection.

Concordance of Culture versus Immunoassay

Among the 83 patients, serum immunoassay analysis was performed on 75 patients and NSA was successfully cultured and assayed for 79 patients. The serum immunoassay detected S. aureus infection in 48 cases (64%), whereas the NSA immunoassay identified S. aureus infection in 50 cases (63.3%). The concordance rate between standard culture and immunoassay for diagnosis of S. aureus were 66% for serum and 61% for NSA medium (Table 2). Among the 45 culture SA+ patients, both serum and NSA immunoassays detected active host immune response against S. aureus infection in 32 cases (71.1%). In 13 (16%) culture SA+ patients, both the serum and NSA immunoassay failed to detect host immune response against S. aureus; suggestive of suspicious false-positive culture or S. aureus not being the dominant infecting pathogen in these cases.

Table 2:

Concordance of immunoassay (Serum and MENSA) versus culture presented in % and number of patients. Gray box represents culture and immunoassay concordant patients and white box represent discordant patients. Abbreviations: MENSA, medium enriched in newly synthesized antibody.

	Immunoassay	Culture	Percentage	Number
Serum	Positive	Positive	43%	32
	Negative	Negative	23%	17
	Positive	Negative	21%	16
	Negative	Positive	13%	10
MENSA	Positive	Positive	41%	32
	Negative	Negative	20%	16
	Positive	Negative	23%	18
	Negative	Positive	16%	13

Immunoassay for Diagnosis of S. aureus Foot Infection

Analysis of serum immunoassay showed significantly higher S. aureus specific antibody titers in the culture SA+ group compared to the culture SA− group; particularly in response to S. aureus antigens SCIN (AUC = 0.68; p = 0.005), ClfB (AUC = 0.64; p =0.02), and IsdH (AUC = 0.63; p = 0.03) (Figure 2A). Although NSA immunoassay showed slightly increased antibodies titers for IsdH (AUC = 0.56; p = 0.36) and LukS-PV (AUC = 0.58; p = 0.22) in culture SA+ group, it did not reach statistical significance compared to culture SA− patients (Figure 2B). Comparative analysis of combined antibodies and culture showed improved concordance in detecting S. aureus infections. In particular, anti-IsdB, ClfB, SCIN (AUC = 0.71, p = 0.0006) and Anti-IsdB, IsdH, SCIN (AUC = 0.71, p = 0.0006) were the best antigen combinations for serum immunoassay (Figure 2C). Whereas, anti-IsdH, LukF-PV, LukS-PV (AUC = 0.66, p = 0.017) and anti-Amd, IsdH, LukF-PV (AUC = 0.63, p = 0.046) were the best three antigen combinations for NSA immunoassay (Figure 2D).

Figure 2.

Correlation of culture versus serum and NSA immunoassay for detection of S. aureus infection using individual and combinations of antigens. Receiver operating characteristic (ROC) curve analysis, summarized by area under the ROC curve (AUC), was used to identify the best individual and combinations of antigens that correlated with S. aureus positive culture. (A) Single antigen analysis of the serum antibodies against SCIN (AUC = 0.68; p = 0.005), ClfB (AUC = 0.64; p =0.02) and IsdH (AUC = 0.63; p = 0.03) showed statistically significant AUCs. (B) Single antigen analysis for NSA medium failed to show an antigen which significantly correlated with culture identification of S. aureus. (C) The AUC levels increased further by combing 3 different antigens for the serum and (D) NSA medium. * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001. Abbreviations: ROC, receiver operating characteristic; AUC, area under the ROC curve; MENSA, medium enriched in newly synthesized antibody.

Immunoassay for Tracking Evolving Infectivity of S. aureus Foot Infection

NSA assay was suggested to have clinical potential for tracking treatment response by accurately evaluating the host’s evolving immune response against S. aureus infection.²⁴ In the case of S. aureus infection resolution with surgical intervention and antibiotics treatment, less stimulation of plasmablast and subsequent decrease in anti-S. aureus antibodies generation is expected in association with clinically healing foot ulcer or surgical incision (Figure 3A). In contrast, persistently elevated NSA against S. aureus antigens was expected in persistent or recurrent infection of the wound (Figure 3B). To test this, the relative changes of NSA titers during postoperative follow-ups compared to that at the initial recruitment time-point was compared to the clinical wound healing status. Participants were categorized by 1) S. aureus infection status as determined by culture (SA+ versus SA−), then 2) wound healing status (healed versus not-healed). Total 45 culture SA+ patients formed the basis of this analysis. Among them, 25 (56%) patients were classified as healed wound and 20 (44%) as not-healed wound.^7,8,24 NSA immunoassay showed significantly greater differences in changes of antibody titers between healed versus not-healed cohorts (Figure 4A). Especially, NSA against IsdH (AUC = 0.74; p = 0.011), LukFPV (AUC = 0.73; p = 0.011), and IsdB (AUC = 0.72; p = 0.014) antigens showed significant correlation with clinical wound healing status (Figure 4B). Combining NSA against IsdH, ClfB, and LukF-PV antigens (AUC = 0.75; p = 0.007) provided the highest correlation with wound healing status (Figure 4C).

Figure 3.

Examples of NSA immunoassays for healed versus not-healed wounds. Each line represents antibodies titers against 8 different S. aureus-specific antigens. (A) NSA immunoassay of S. aureus infected DFU patient who healed the wound. NSA immunoassay showed relatively high titers at 0 weeks, which decreased at 8 weeks, and remained low at the final 12 weeks follow-up. (B) Immunoassay of a S. aureus infected DFU patient who failed to heal. NSA immunoassay showed decreased titers up to 8 weeks, then elevated at 12 weeks as recurrent infection occurred. Abbreviations: DFU, diabetic foot ulcer; MFI, mean fluorescence intensity; MENSA, medium enriched in newly synthesized antibody.

Figure 4.

Correlation of wound healing status versus evolving antibody titers using individual and combinations of antigens. S. aureus positive patients, identified by culture, were divided as Healed versus Not-healed groups at the final follow-up. (A) The anti-S. aureus antibody titers changes (initial to final follow-up) for 8 different antigens were calculated for serum (Serum SA+) and NSA (MENSA SA+). Then the ratios of antibody titers changes in Not-healed/Healed groups were calculated. The difference in titers of Not-healed/Healed is much greater for NSA medium (MENSA SA+) compared to serum. This indicates NSA titer changes showed stronger correlation with wound healing status. (B) The single antigen ROC curves were calculated to investigate correlation between NSA antibody titers changes versus clinical wound healing status. IsdH (0.74; p = 0.011), LukF-PV(0.73; p = 0.011) and IsdB (0.72; p = 0.014) were significantly correlated with healing status. (C): AUCs analysis using combinations of 3 antigens revealed multiple combinations which could accurately track wound healing status. The IsdH, ClfB, and LukF-PV antigens combination showed the highest AUC value of 0.75 (p = 0.007). * indicates p < 0.05. Abbreviations: SA+, S. aureus positive culture; SA−, S. aureus negative culture; ROC, receiver operating characteristic; AUC, area under the ROC curve; MENSA, medium enriched in newly synthesized antibody.

DISCUSSION

There is a great demand for accurate identification of the dominant pathogen in polymicrobial infections to deliver an effective post-operative antibiotic treatment. Standard microbial culture identification of pathogens has been reported to be inaccurate and its semiquantitative measure does not accurately reflect pathogenicity.^7,24,35–37 This inaccuracy and lack of specificity further complicates effective treatment of the main pathogen in polymicrobial foot infection. In this work, we aimed to investigate the use of a S. aureus specific immunoassay in identifying the dominant pathogen among multiple microorganisms identified by culture. We demonstrated that this S. aureus-specific immunoassay can supplement standard microbial culture to identify the main pathogen and monitor host’s immune response, using both serum and NSA, which reflects evolving infectivity during the postoperative period. This assay may be capable of identifying false-positive or false-negative culture growth and detect a persistent or recurrent infection. Since S. aureus is the most common pathogen (46-68%) in DFU, ^{7,8,12,16,17,22–25} we focused our current investigation to this species. Future work will expand on this study by investigating other species commonly infecting the foot.

Here we show that the concordance rate between bone culture and immunoassay ranged from 61% (NSA) to 66% (Serum). Although serum and NSA assay showed higher detection rates of S. aureus infection compared to standard culture, we are unable to infer that our immunoassays were more sensitive than culture without a tertiary diagnostic method. However, it raises a suspicion for false-negative growth in clinically infected patients. This finding suggests that immunoassays may detect culture-negative S. aureus infections that warrant further investigation with molecular diagnostics, such as 16S rRNA sequencing analysis or Staphylococcal Protein A (spa) genotyping to confirm S. aureus infections.^{10,24,35–37} A subset (26 patients) of the current study cohort in which spa genotyping was possible revealed a 85% concordance rate with the immunoassay.²⁴ Without a tertiary method of infective species identification for all patients, the exact sensitivity of our immunoassays cannot be concluded. Some cultures may grow a contaminant or commensal and yields false-positive growth, whereas some patients may have been on antibiotics prior to surgery, resulting in false-negative culture. Discrepancy in culture versus immunoassay may be clarified further by comparing with a tertiary molecular diagnostic method, such as next generation sequencing (NGS).^35–37 Another notably attribute of diagnostic immunoassay may relate to cost-effectiveness. Its cost is estimated to be $200, in contrast to $60 per sample for culture.

In this study, the serum immunoassay was superior to NSA in initial diagnosis of S. aureus infection (Figure 2A & B). This result may be due to analytic sensitivity or efficient capture of plasmablasts prior to sensitization. Combining three antigens substantially improved the concordance rates between the culture and both serum and NSA immunoassays in detecting S. aureus infections (Figure 2C & D). The application of serum immunoassay for species-specific diagnosis of orthopaedic infections has been reported and commercialized. Marmor et al utilized a serum multiplex immunoassay to successfully diagnose S. aureus, S. epidermidis, S. lugdunensis, S. agalactiae and Propionibacterium acnes in hip, knee and shoulder prosthetic joint infections.¹⁹ However, since their immunoassay utilized only the serum the application was limited to identification of pathogens without the ability to track evolving pathogenicity.

Commensal S. aureus in human skin has reported to elicit both innate and adaptive immunity, which leads to high-titers of anti-S. aureus antibodies for years in even healthy individuals.⁴⁰ Therefore, this characteristic precludes serum-based assay from being a promising tool for tracking treatment response despite its clinical utility to diagnose new S. aureus infections in DFUs. Since ASCs are only secreted during an ongoing, active infection, NSA immunoassay is a promising method for tracking antibiotic treatment response and detecting recurrent infections.^{9,13,14,20,22,24} Our results showed that the NSA titers changes during postoperative period against various S. aureus antigens were significantly correlated with wound healing status (Figure 4). The conventional inflammatory markers, such as ESR and CRP, are not specific to a pathogen, disease, or site of infection and may not accurately represent evolving infection. In our pilot study of 26 infected DFU patients, 10 were confirmed to have S. aureus infections. Among the 10 patients, 6 patients healed the DFU, whereas 4 did not. We noted remarkable changes in NSA immunoassay over time significantly reflected the healing status. In some who failed to heal, we noted borderline inflammatory markers (CRP or ESR) value, but resurging anti-S. aureus antibody levels at their follow-ups.²⁴ Combination of borderline inflammatory markers value with ambiguous clinical picture of indolent or recurrent infection may present a diagnostic dilemma to surgeons. In such case, this novel immunoassay may provide further diagnostic and prognostic insight to guide further treatment.

There are a number of limitations in this study. First, we only investigated S. aureus infection in polymicrobial DFU. This novel diagnostic method can be expanded to include other species, such as S. agalactiae, S. epidermidis, and pseudomonas aeruginosa, which commonly infects DFU.^2,7,8,16,17 Second, most diabetic patients have various other medical comorbidities and biomechanical derangements, which may have affected their wound healing process. To mitigate this effect, we excluded patients with vascular insufficiency and evidence of immune deficiency or malnutrition. Whenever possible, we tried to address biomechanical derangement of the foot through surgical or conservative means as described. However, it is challenging to account for various other medical and biomechanical factors that could have negatively affected wound healing process. Third, patients were referred to the surgeon’s office at various stages of infection, which may have affected their elicited immune response. Some of the participants were on oral antibiotics prior to recruitment, which may have confounded the culture or immunoassay results. Finally, the immunoassay was compared only against standard culture for assessing its ability to diagnose S. aureus infection. Since standard culture is prone to error, a tertiary diagnostic test with higher sensitivity and resolution, such as emerging molecular diagnostics like NGS may have served as a better counterpart to compare against.^18,35–37 Despite the above limitations, the current study suggests a potential for clinical application of immunoassay for species-specific diagnosis and management of foot infection.

Conclusion

This species-specific immunoassay may supplement standard culture for identification of S. aureus infection in a polymicrobial foot infection. It was also able to identify 21-23% potentially false-negative cultures in DFU patients that subsequently needed therapy for S. aureus infections. Moreover, NSA immunoassay can track evolving pathogenicity in response to treatment and detect recurrent infection. Overall, this novel method can help orthopaedic surgeons identify the infecting pathogen, detect recurrent infection, and promptly guide treatment intervention.