Tracking Anti-Staphylococcus aureus Antibodies Produced In Vivo and Ex Vivo during Foot Salvage Therapy for Diabetic Foot Infections Reveals Prognostic Insights
Gowrishankar Muthukrishnan et al. 
E-mail: gowri_shankar@urmc.rochester.edu

INTRODUCTION: The growing prevalence of type 2 diabetes mellitus (DM) in the United States and world populations has led to the steadily increasing frequency of its associated sequelae, including diabetic foot infections (DFI). Recent assessments estimate that as many as 26% of Americans over the age of 65 years have Type 2 DM.  The cost associated with management of DM and its associated ailments is a significant burden on the U.S. healthcare system1,2. In the DM patient population, the risk of developing a foot ulcer is 15% with two-thirds of lower extremity amputations associated with DFI.  Moreover, the five-year mortality rate of DFI has been reported to be 50%; a mortality rate equal to the most life-threatening of cancers3,4. Management of foot salvage therapy (FST) for DFI is challenging due to the absence of reliable diagnostics to identify the etiologic agent, and prognostics to justify aggressive treatments. Here, we introduce a highly sensitive multiplex immunoassay capable of tracking anti-S. aureus antibodies in vivo and ex vivo, for diagnosing S. aureus infections in DFI patients, and prognosing the success or failure of their FST.

METHODS: 
Patient Enrollment: The current study was approved by the University of Rochester Medical Center’s research subjects review board (RSRB#00057719). Twenty-six (26) patients presenting with DFI and eligible for FST were enrolled after obtaining informed consent. There were 21 males and 5 females with the average age of 58 (range; 46.3 to 69.7) years and average BMI of 32.5 ± 6.3. All patients underwent surgical debridement, followed by collection of soft tissue and bone samples for identification of the infecting pathogen via 16S rRNA sequencing and Staphylococcal protein A (spa) genotyping5,6. 
Healing versus Non-Healing Classification: For each subject, wound size was measured, and depth was classified according to Wagner’s classification7. Healing wound was defined as completely healed or decreased size and wound depth, while non-healing (NH) wound is defined as unimproved or increased size and depth with persistent 12 signs and symptoms of infection7. Multiplex Luminex Immunoassay: Whole blood was obtained over 12 weeks of FST, and purified peripheral blood mononuclear cells were cultured to produce medium enriched for newly synthesized anti-S. aureus antibodies (MENSA). Anti-S. aureus IgG levels in sera and MENSA were measured using a custom Luminex-based multiplex immunoassays developed by our group8. 
Statistical Analysis: Antibody measurements obtained from serum and MENSA were assessed for predictive ability in discriminating infection status and healing status using receiver operating characteristic (ROC) curve analysis, with overall accuracy summarized by the area under the ROC curve (AUC). P-values for testing the significance of each AUC, and for comparing AUC's across predictors, were also computed.

RESULTS: Seventeen out of 26 infections were polymicrobial, and 12 were positive for S. aureus. While antibody titers in serum and MENSA displayed similar diagnostic potential to detect S. aureus infection, MENSA showed a two-fold greater signal to background ratio. Multivariate analyses revealed increases in predictive power of diagnosing S. aureus infections (AUC > 0.85) when combining titers against multiple antigens. The greatest combination in serum was IsdB+FnBPA+ClfB+LukF-PV (AUC = 0.9697, ***p < 0.001), and the greatest combination in MENSA was IsdA+CHIPS+IsaA+PSM (AUC = 0.9306, ***p < 0.001). We also assessed the prognostic potential of our serum and MENSA immunoassays for their ability to track the success or failure of FST. Remarkably, anti-S. aureus IgG levels in MENSA decreased with successful FST, and rose with re-infection, while, IgG levels in serum remained unchanged throughout the 12-week FST.

DISCUSSION: The strength of our blood-borne approach is in the detection of pathogens that actually cause infection and activate the immune response. The novel analytic fluid, MENSA, can also accurately monitor treatment response and detect persistent or recurrent infection. However, this proof of concept study is preliminary due to small sample size, and a larger cohort is required to achieve greater statistical power. Additionally, MENSA-based clinical testing may require a better understanding of the timing of an ongoing infection’s immune response. Most importantly, true clinical utility for the proposed analytical approach will require the ability to detect and quantitate all bacteria in these polymicrobial infections, including S. aureus, S. epidermidis, S. agalactiae, Group B Streptococcus and Enterobacteriae.

SIGNIFICANCE: We demonstrated the potential for two innovative analytic approaches for diagnosing and tracking S. aureus infections in DFI patients undergoing FST: 1) A novel analytic fluid, MENSA, for tracking rapid changes in the immune response and 2) Multiplex immunoassays to identify infecting pathogens in a polymicrobial infected open wound in DFI patients.

REFERENCES: 1) Armstrong DG et al. N Engl J Med (2017) 376:2367-2375. 2) Gregg EW et al. (2018) Lancet 391:2430-2440. 3) Robbins JM et al. (2008) J Am Podiatr Med Assoc 98:489-93. 4) Hicks CW et al. (2016) Ann Vasc Surg 33:149-58. 5) Shopsin B et al. J Clin Microbiol 37:3556-63. 6) Harmsen D et al. J Clin Microbiol 41:5442-8. 7) Gardner SE et al. Diabetes 62:923-30. 8) Nishitani K et al. Clin Orthop Relat Res 473:2735-49.

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