These results extend our previous work on neonatal stool, and the work of others on stools from subjects with bacterial enteric infections, by portraying the ability to characterize the host inflammatory response to these pathogens using fecal mRNA analysis. We also demonstrate the variable correlation of fecal mRNA abundance with the cognate protein. This disparity was most notable for calprotectin mRNA abundances and protein antigen.
We cannot state if these results are specific for infectious injuries to the gut, and/or the extent to which specific mRNAs might be found in non-infectious gastrointestinal inflammation. Stool cytokines (usually detected via immunoassays) are often elevated in inflammatory bowel disease,14,15
so it would not be surprising if corresponding fecal mRNAs are also present in these and other inflammatory disorders and elevated compared to controls. It is also possible that additional colonic (e.g., diverticulitis, ischemic colitis) and more proximal disorders (e.g., celiac disease) might also be characterized by measuring stool mRNAs.
Target stability is a major consideration in any assay, and analyses of molecules in stool present particular concern. We have noticed proteolysis in stools using spiked cytokines and protein immunoblots (data not shown), and it is possible that host or microbial proteases render protein molecules undetectable by EIA, either because of degradation or denaturation of target. There could be additional reasons for non-detection of calprotectin by EIA beyond proteolysis. The EIA kit we employed uses antibodies to the calprotectin complex (a heterodimer of S100A8 and S100A9 subunits), while the primers used to detect calprotectin via qRT-PCR were for only the S100A8 moiety. Denaturation of the protein or dissociation of the subunits could hinder EIA detection if the target epitopes are changed or separated.
We previously demonstrated the relative stability of host mRNA in neonatal stool.5
Even if RNA degradation does occur in stool, as long as the mRNA of interest and the GAPDH transcripts degrade at the same rates, and sufficient non-degraded transcript (the calprotectin and GAPDH primers produce RNA amplicons that are 107 and 122 base pairs, respectively) remains, ratios can be determined. There are two additional advantages to determining mRNA abundance in stool, compared to assays of proteins. First, mRNAs are normalized to a host housekeeping transcript, and are independent of fecal water content. This is particularly important in diarrhea, where dilutions of target protein could yield falsely low values in patients compared to controls with solid stools. Second, mRNA detection is more economical. By our calculations, the reagent cost of performing duplicate EIA determinations on a specimen is about twice that of assessing relative mRNA abundances. Indeed, the per-assay cost differences are probably greater when considering that the fixed cost of the RNA preparation, which accounts for about half the cost of a single mRNA abundance determination, can be offset by the ability to measure multiple additional transcripts. Such single analyte multidimensional utility increases the ability to profile digestive organ response with parsimonious use of reagents.
In summary, we extend previous work by demonstrating that human stool mRNAs are quantifiable analytes in infectious colitis. mRNAs from several proteins important in inflammatory cascades are consistently elevated in the stools of patients with acute infectious colitis, and such assessments provide greater sensitivity than immunoassays. To the extent we tested, there is no evidence that EIAs better detect inflammation than mRNA abundances, and it appears likely that relative mRNA abundances offer equivalent or improved sensitivity for characterizing host response in acute bacterial enteric infections.