Nasopharyngeal viral infection as a source of elevated fecal calprotectin: a diagnostic pitfall in intestinal inflammation

Fecal calprotectin is widely regarded as a sensitive and specific biomarker for detecting intestinal inflammation, and it plays a pivotal role in the clinical management of IBD. It is primarily used to guide clinical decision-making in IBD, and elevated levels can trigger downstream consequences such as unnecessary treatment escalation or invasive investigations if misinterpreted. Because calprotectin levels correlate with neutrophilic infiltration of the intestinal mucosa, the test has become a mainstay in the non-invasive diagnosis and monitoring of IBD. Its use is now firmly embedded in both clinical trials [1]and routine medical practice [2], where it supports the assessment of disease activity, treatment response, and early prediction of relapse without immediate recourse to invasive endoscopic procedures. Elevated fecal calprotectin is typically interpreted as bona fide evidence of gastrointestinal inflammation. For instance, in patients suffering from diarrhea due to SARS-CoV-2 infection, elevated fecal calprotectin levels have been accepted as strong evidence that direct infection of the intestinal epithelium plays a central role in the pathogenesis of this condition [3]. However, non-intestinal sources of calprotectin, such as Helicobacter pylori infection, have also been reported to elevate fecal calprotectin, which acts as a potential confounder [4‐6].

Biochemically, calprotectin is a heterodimeric complex of the S100 family of calcium-binding proteins—specifically S100A8 and S100A9—which are presumed to be released predominantly by activated neutrophils and macrophages during acute inflammation [7]. It also exists as a stable, biologically active heterotetrametric, which may contribute to its persistence and detectability in feces. Hence, its fecal concentration is thought to reflect the activity of these innate immune cells within the intestinal mucosa. Nonetheless, over the years, a partial disconnect has been observed between endoscopic inflammation and fecal calprotectin levels, raising the possibility that calprotectin might also originate from non-intestinal or non-phagocytic sources, a concept that has received relatively little attention in the literature [8].

Intriguingly, and in a different biological context, we have previously observed that upper gastrointestinal squamous epithelia, particularly in the esophagus, express high levels of calprotectin [9]. This expression appears to be constitutive and not necessarily inflammation-driven. Transcriptomic, proteomic, and immunohistochemical data was analyzed, specified in the material methods section under the bioinformatics analysis of online database. This analysis revealed that the subunits S100A8 and S100A9 are highly expressed in rostral epithelia, including the nasopharyngeal, oropharyngeal, and esophageal linings, whereas caudal gut segments such as the ileum and colon show much lower baseline expression levels (Fig. 1A). Based on these observations, we hypothesized that damage to proximal endodermal epithelium, for example due to upper respiratory tract infections, might release calprotectin from these tissues into the gastrointestinal tract. This calprotectin could then survive passage through the gut and be detected in feces—producing elevated fecal calprotectin levels that are not indicative of intestinal mucosal inflammation but still misinterpreted as such (see model in Fig. 1B).

In order to assess the resistance of calprotectin to proteolytic degradation and chemical breakdown during transit through the gastric and intestinal environments, we conducted an ingestion study using porcine-derived calprotectin, which shares structural homology with human calprotectin but can be specifically detected using a pig-specific ELISA (Cusabio, CSB-Eq. 013485PI) [10, 7]. In a controlled experiment, healthy volunteers (n = 4) ingested a defined quantity of epithelial porcine calprotectin. Fecal samples collected before and after ingestion were assayed using the species-specific ELISA. The results demonstrated a significant and reproducible increase in fecal porcine calprotectin following ingestion (p < 0.01; Fig. 1C), providing direct experimental evidence that exogenous calprotectin from proximal epithelial sources can survive gastrointestinal transit and be recovered in the feces.

To further investigate the relevance of this mechanism under pathophysiological conditions, we performed a retrospective cohort study involving patients who had undergone nasopharyngeal swabbing and tested positive for either SARS-CoV-2 (n = 38) or non-SARS-CoV-2 respiratory viruses (n = 20). Fecal calprotectin levels were measured and compared between these groups. Interestingly, patients with non-SARS-CoV-2 respiratory viral infections exhibited markedly higher fecal calprotectin levels compared to COVID-19 patients (447 µg/g vs. 82 µg/g, respectively; p < 0.001; Fig. 1D), despite the absence of gastrointestinal symptoms in many cases.

Quantification of calprotectin in nasopharyngeal swabs from the same patients revealed no significant difference in calprotectin concentration between the SARS-CoV-2 and non-SARS-CoV-2 groups (Fig. 1E), suggesting that nasopharyngeal calprotectin levels per se are not virus-specific. Notably, no correlation between swab and fecal calprotectin was observed in the non-SARS-CoV-2 group, likely due to a smaller sample size, greater heterogeneity, and additional biological factors driving the elevated fecal calprotectin values in this group which were high in comparison to the values in the SARS-CoV-2 group (Fig. S1). Within the COVID-19 cohort, a positive correlation was observed between swab calprotectin levels and fecal calprotectin (R = 0.43; p = 0.008; Fig. 1F), indicating a potential dose-dependent relationship between epithelial calprotectin release and its gastrointestinal detection. Furthermore, fecal calprotectin levels in COVID-19 patients were found to correlate positively with viral load in the nasopharynx, as reflected by RT-PCR cycle threshold (Ct) values (R = 0.13; p = 0.0257; Fig. 1G). These findings support the view that nasopharyngeal viral activity contributes to systemic or luminal calprotectin levels and may represent a confounding factor in interpreting fecal calprotectin as a marker for gut inflammation.

Collectively, these results provide compelling evidence that upper respiratory tract viral infections can be a significant non-intestinal source of elevated fecal calprotectin. This has critical implications for clinical practice. Although the sensitivity of fecal calprotectin for detecting intestinal inflammation is well-established and approaches 100% in many studies [11], its specificity is known to be more variable [12]. Indeed, elevated calprotectin levels can also be seen in infectious enteritis, colorectal cancer, NSAID enteropathy, and—as shown here—extraintestinal infections. Despite widespread use of fecal calprotectin in clinical algorithms for IBD, the potential for false-positive results arising from non-intestinal sources has not been adequately addressed. Clinically, fecal calprotectin values above 250 µg/g are generally considered to indicate active intestinal inflammation, whereas results in the 100–250 µg/g “grey zone” often prompt repeat testing or further investigation [13]. Our data suggest that patients with upper respiratory viral infections may fall into this intermediate range or even exceed diagnostic thresholds, creating potential for false-positive interpretations. Such misinterpretation could lead to unnecessary endoscopic evaluation, escalation of therapy, or increased patient anxiety in the absence of true intestinal pathology. Our findings indicate that clinicians should exercise greater caution in interpreting elevated fecal calprotectin levels, particularly in patients who lack other clinical indicators of intestinal inflammation. Rather than recommending routine virological testing, a more appropriate approach may be to ask patients if symptoms of viral infections had occurred surrounding the moment of sampling. Subsequently, to re-test fecal calprotectin after resolution of upper respiratory symptoms, thereby reducing unnecessary invasive diagnostic or therapeutic interventions. The observed variability in non–SARS-CoV-2 viral cases likely reflects differences in viral tropism, epithelial involvement, and host inflammatory responses among pathogens, underscoring the broader limitation that the impact of upper respiratory tract infections on fecal calprotectin levels remains insufficiently understood. While we demonstrate a clear association, the extent to which different viral pathogens, infection severity, and host-related factors influence this phenomenon has not yet been established. Larger prospective studies will be needed to further delineate the scope and clinical implications of this effect. This work therefore expands our understanding of the biological origins of fecal calprotectin, demonstrating that calprotectin derived from proximal epithelial tissues—either ingested or released due to nasopharyngeal viral infection—can transmit the gastrointestinal tract and be detected in feces. Recognizing this phenomenon is essential for avoiding misdiagnosis and unnecessary procedures in patients with elevated fecal calprotectin but no other compelling clinical evidence of intestinal inflammation. Future diagnostic protocols may benefit from incorporating virological screening or serological markers to help distinguish genuine intestinal inflammation from systemic or proximal epithelial sources of calprotectin. As we refine the role of this biomarker in gastrointestinal medicine, contextual interpretation becomes key to ensuring its proper use in guiding clinical decisions.