Novel Immunoassay for Diagnosis of OngoingClostridioides difficileInfections Using Serum and Medium Enriched for Newly Synthesized Antibodies (MENSA)

Abstract

47BACKGROUND. Clostridioides difficile infections (CDI) have been a challenging and 48 increasing serious concern in recent years. While early and accurate diagnosis is crucial, available assays 49 have frustrating limitations 50 51 OBJECTIVE. Develop a simple, blood-based immunoassay to accurately diagnose patients 52 suffering from active CDI. 53 54 MATERIALS AND METHODS. Uninfected controls (n=95) and CDI patients (n=167) were 55 recruited from Atlanta area hospitals. Blood samples were collected from patients within twelve days of a 56 positive CDI test and processed to yield serum and PBMCs cultured to yield medium enriched for newly 57 synthesized antibodies (MENSA). Multiplex immunoassays measured Ig responses to ten recombinant C. 58 difficile antigens. 59 60 RESULTS. Sixty-six percent of CDI patients produced measurable responses to C. difficile 61 antigens in their serum or MENSA within twelve days of a positive CDI test. Fifty-two of the 167 CDI 62 patients (31%) were detectable in both serum and MENSA, but 32/167 (19%) were detectable only in 63 MENSA, and 27/167 (16%) were detectable only in serum. 64 65 DISCUSSION. We describe the results of a multiplex immunoassay for the diagnosis of ongoing 66 CDI in hospitalized patients. Our assay resolved patients into four categories: MENSA-positive only, 67 serum-positive only, MENSA-and serum-positive, and MENSA-and serum-negative. The MENSA 68 positive-only patients accounted for 30% and may be attributed to nascent antibody secretion in MENSA 69 prior to seroconversion. Conversely, the serum positive-only subset may have been more advanced in 70 5 5 their disease course. Immunocompromise and misdiagnosis may have contributed to the 34% of CDI 71 patients who were not identified using MENSA or serum immunoassays. 72 73 IMPORTANCE. While there was considerable overlap between patients identified through 74 MENSA and serum, both methods detected additional, unique patients. The combined use of both 75 MENSA and serum to detect CDI patients resulted in the greatest identification of CDI patients. Together, 76 longitudinal analysis of MENSA and serum will provide a more accurate evaluation of successful host 77 humoral immune responses in CDI patients. 78In this observational investigation, we present the results of a pilot study validating the use of a 127 multi-antigen immunoassay and its ability to measure antibody secretion reflecting ongoing CDI in 128 MENSA samples from a complex hospital population and compare its performance with results from sera. 129 130 RESULTS 131

Relaxed peripheral tolerance drives broad de novo autoreactivity in severe COVID-19

“…Previous work has correlated expansion of the ASC compartment correlated with increased serological IgG responses to the SARS-CoV-2 spike protein receptor binding domain (RBD) in patients with severe disease, however it did not directly test antigen specificity 8 . Using a novel in vitro culture method, ASCs from the ICU-C and OUT-C cohorts were cultured overnight and the supernatant, or media enriched in newly synthesized antibodies (MENSA) was collected 20 . MENSA analysis, which is more specific to the circulating ASC compartment, revealed an increased cellular component in the blood of these ICU-C patients capable of secreting IgG RBD-specific antibodies, and confirming the relevance of early circulating ASCs to antiviral response as opposed to non-specific cellular expansion (Fig 1e).…”