Absolute versus relative presentation of tcdB Clostridium difficile biomarker

Abstract

Clostridium difficile infection is the major etiologic agent of antibiotic-associated pseudomembranous colitis, a disease that can be fatal if unrecognized, or untreated. On average, there are 15000 deaths and 500000 new cases per year, in the USA. Diagnostic biomarkers currently used are the tcdB gene, or its gene product (toxin B). Clinical interpretation of the assay is particularly challenging: (1) biomarker detection is possible without manifestation of symptoms and (2) missing biomarker due to assay sensitivity limitations can be fatal. To resolve clinical uncertainty, quantitative analysis has been considered. Despite multiple efforts, a quantitative tcdB/toxB threshold with a meaningful clinical impact has yet to be established. Herein we shed light as to why mass/volume-based normalisations were fruitless in the past. Specifically, measuring total bacterial flora (using "universal bacterial" 16S qPCR rDNA assay) to calculate relative abundance of C difficile, we demonstrate a strong and significant negative correlation between tcdB biomarker of C difficile (R 2 =0.73, N=227, P=10 -39 ) and the rest of gut microorganisms. The new parameter (Cq (toxB)-Cq (16SrDNA)) is calculated from two biomarkers, is independent of sampling variability and inherently incorporates the destructive character of C difficile on the rest of micro-flora. By incorporating relative abundance of tcdB (in context of the total bacterial flora), and correcting for "biomass wash-out/dilution effects of acute diarrhea , these biomarkers could collectively enhance the predictive value of CDI testing.