Circulating Calprotectin as a Potential Biomarker of Persistent Olfactory Dysfunctions in Post-COVID-19 Patients

doi:10.21203/rs.3.rs-3332286/v1

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Research Article

Circulating Calprotectin as a Potential Biomarker of Persistent Olfactory Dysfunctions in Post-COVID-19 Patients

https://doi.org/10.21203/rs.3.rs-3332286/v1

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published 01 Sep, 2024

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Objective and Design:

This longitudinal prospective study aims to investigate the potential of circulating calprotectin (cCLP) as a biomarker in persistent olfactory dysfunctions following COVID-19 infection.

Materials and Methods

Thirty-six patients with persistent hyposmia or anosmia post COVID-19 were enrolled (HT0) and re-evaluated after three months of olfactory training (HT1). Two control groups included 18 subjects without olfactory defects post COVID-19 (CG1) and 18 healthy individuals (CG2). Nasal and blood samples were collected to assess circulating calprotectin levels.

Results

Higher calprotectin levels were observed in serum and nasal supernatant of hyposmic patients (HT0) compared to control groups (CG1 and CG2). Post-olfactory training (HT1), olfactory function improved significantly, paralleled by decreased calprotectin levels in serum and nasal samples.

Conclusions

Circulating calprotectin holds potential as a biomarker in persistent olfactory dysfunctions after COVID-19. The decrease in calprotectin levels post-olfactory training implies a role in monitoring and evaluating treatment responses. These findings contribute to the growing literature on potential biomarkers in post-COVID-19 olfactory dysfunctions and underscore the importance of investigating novel biomarkers for personalized patient management. Nevertheless, further studies are needed to validate the application of calprotectin assay in nasal diseases and its correlation with nasal cytology.

Calprotectin

Long COVID

Hyposmia

Olfaction

SARS-CoV-2

Calprotectin (CLP) is a calcium and zinc-finger heterodimer formed by proteins S100A8 and S100A9, known by various synonyms such as leukocyte protein L1, myeloid-related proteins-8 and − 9 (MRP8, MRP9), and calgranulin (A and B); it plays a crucial role in innate immunity and inflammatory processes[1–3]. Neutrophils are the primary producers and releasers of CLP, accounting for up to 40% of the granules’ cytosolic proteins[4]. Calprotectin is also constitutively expressed by dendritic cells, monocytes, activated macrophages, oral keratinocytes and squamous mucosal epithelium[1, 4].

Upon encountering pro-inflammatory stimuli, granulocytes rapidly increase CLP expression and secretion, leading to its multifaceted activities[5, 6]. First and foremost, CLP acts as an endogenous ligand of Toll-like receptor 4 (TLR4) and the receptor of advanced glycation end-products (RAGE), inducing a cascade of proinflammatory responses. This includes NF-κB/MAPK activation, which in turn promotes intracellular signalling cascades, the induction of cytokines and chemokines, and recruitment of inflammatory cells. Therefore, CLP acts as a cytokine-like protein by binding surface receptors and triggering pathways engaged in mounting immune response[7]. Calprotectin exhibits potent antimicrobial properties by sequestering zinc and inhibiting zinc-dependent enzymes, a mechanism employed to prevent bacterial growth and exert fungistatic functions. Additionally, CLP serves as a danger-associated molecular pattern (DAMP) in sterile inflammation, contributing to host defense mechanisms[8, 9].

Notably, in the gastrointestinal tract, where it is primarily produced and released by neutrophils, fecal CLP has become widely used as a non-invasive marker to assess disease activity and monitor treatment response in inflammatory bowel diseases (IBD), such as Crohn's disease and ulcerative colitis[10–12]. In fact, CLP levels in feces are typically around six times higher in healthy subjects compared to normal plasma levels. During inflammation, these levels can increase dramatically, up to 100 times higher, making fecal CLP (fCLP) a reliable biomarker[13].

In recent years, due to its ability to reflect inflammation and tissue damage, circulating CLP (cCLP) has garnered significant attention and has also emerged as a potential biomarker in several clinical conditions[8, 14–17]. Studies have suggested its utility as a biomarker of disease activity in autoimmune rheumatic diseases, cystic fibrosis, chronic asthma, and different phenotypes of chronic rhinosinusitis with nasal polyps (CRSwNP)[18–21]. To note, CRSwNP frequently presents with olfactory dysfunctions, which appear to be closely correlated with the local inflammatory environment and clinical-cytological grading[22].

In the context of COVID-19 pandemic, circulating CLP has been recognized as a hallmark of disease severity, with higher levels observed in cases requiring mechanical ventilation during SARS-CoV-2 acute infection[23–27]. Moreover, the presence of anti-calprotectin antibodies has shown potential as a predictor of a favourable outcome in post-acute sequelae of SARS-CoV-2 infection (PASC), hinting at their possible protective role in the pathology of various forms of PASC[28, 29].

Remarkably, SARS-CoV-2 is among the viruses frequently associated with post-viral olfactory disorders (PVOD), primarily invading the olfactory neuroepithelium through the angiotensin-converting enzyme II (ACE2) receptor and transmembrane protease serine enzyme (TMPRSS2)[30]. While olfactory alterations typically resolve within weeks after SARS-CoV-2 infection, up to 30% of individuals develop persistent olfactory dysfunctions as part of PASC, lasting at least four weeks from the initial infection. Long-term olfactory defects have been linked to downregulation of receptor proteins, suggesting that persistent damage to ciliate olfactory receptor cells, sustentacular cells, and olfactory ensheathing cells could potentially play a role in the establishment of olfactory dysfunctions [31–33]. Among the others, olfactory ensheathing cells play a crucial role for supporting the regeneration of olfactory neuron axons [34, 35], and expressing ciliary neurotrophic factor (CNTF), which promotes the proliferation of olfactory stem cell lineage [36].

Nevertheless, the pathophysiology underpinning olfactory dysfunctions in PASC patients remains to be fully elucidated. In some cases, damage may manifest independently or simultaneously at different levels of the olfactory pathway, from the sensory epithelium in the olfactory fossa to the olfactory bulbs and cerebral cortex[37–39]. A possible explanation lies in the persistent nasal inflammatory microenvironment following the primary SARS-CoV-2 infection[31, 33].

Despite its potential significance, studies investigating the role of CLP in different forms of PASC, particularly in long-lasting olfactory alterations after SARS-CoV-2 infection, are currently limited. Considering the recognition of circulating calprotectin as valuable biomarker in both COVID-19 and chronic rhinosinusitis with nasal polyps associated with olfactory defects, our study aims to investigate the systemic production of circulating calprotectin in serum, as well as its local production in nasal fluids, in PASC patients with long-term persistent olfactory dysfunctions after SARS-CoV-2 infection. Calprotectin levels have been assessed before and after three months of olfactory training (OT) to gain valuable insights into its association with the severity and progression of olfactory dysfunction post-COVID-19.

2.1. Study design and population

A longitudinal prospective study was conducted at the Otorhinolaryngology Department of University of Rome Tor Vergata, from April 2022 to October 2022. A total of 36 patients affected by PASC persistent olfactory dysfunctions 3–18 months after COVID-19 were enrolled (HT0). This group was tested again after 3 months of an olfactory training protocol (HT1). Besides, two control groups each consisting of healthy subjects were tested, subdivided as follows: 18 subjects without olfactory defects 3–18 months after SARS-CoV-2 infection (CG1); 18 subjects never infected by SARS-CoV-2, without long-lasting olfactory alterations (CG2). The groups of patients were made homogeneous for gender and age (HT0 and HT1 mean age: 52 years; CG1 mean age: 46 years; CG2 mean age: 45 years).

Written informed consent was collected from all participants. Policlinico Tor Vergata Ethical Committee approval was sought and obtained (protocol number 9962/2022).

2.2. Clinical evaluation

Each involved subject in the present study underwent a nasal endoscopy using a 0 degrees angle, 15 cm length and 3.0 mm diameter rigid videoendoscope (Karl Storz GmbH, Tuttlingen, Germany) to assess Lund-Kennedy score [40].

To evaluate olfactory function, we conducted the Sniffin' Sticks extended test n-Butanol, using the "Sniffin’ Test" kit (Burghart, Medizintechnik, GmbH, Wedel, Germany). This widely used olfactory test in Europe is a chemosensory nasal test based on pen-like odor dispensing devices[41]. The test comprises three different subtests: odor threshold, odor discrimination, and odor identification [42]. Felt-tip pens, filled with liquid or odorants dissolved in propylene glycol (total volume of 4 ml), were used for smell presentation. The pens' tip was placed approximately 2 cm from the nasal brim, with each nasal cavity receiving the smell presentation for about 3 seconds, and a 30-second interval between presentations was maintained to prevent olfactory desensitization.

The identification subtest consisted of 16 common odors meeting specific criteria: subjects’ familiarity; same intensity and hedonic tone, and correct recognition by at least 75% of healthy subjects from a list of four different options. The discrimination subtest comprised 16 odorant pen-tip triplets, where patients were asked to recognize the different one. The threshold subtest included 16 odorant triplets with varying degrees of n-butanol dilution, aiming to detect the least diluted odorant in two successive presentations. All these olfactory measurements were conducted in quiet, well-ventilated rooms.

The results of the three subtests were combined to create a "TDI score", resulting from the measures of Threshold, Discrimination and Identification data, representing the overall olfactory function. A score of ≥ 31 indicated normosmia; a score between 31 and 17 indicated hyposmia; a score < 17 defined anosmia.

Furthermore, quality of life was evaluated using the brief version of the Questionnaire of Olfactory Disorders (brief QOD)[43]. This questionnaire consists of 14 items related to patient-reported outcomes in olfactory-specific quality of life (QOD-QOL, 7 items), parosmia (QOD-P, 4 items) and 3 different visual analogue scales (QOD-VAS). QOD-QOL and QOD-P items were scored on a scale from 0 to 3, reflecting the degree of agreement with the statement. The QOD-P and QOD-QOL scores ranged from 0 to 12 and 0 to 21, respectively. Regarding QOD-VAS, each item score ranged from 0 to 10, where 0 indicated "not at all" and 10 indicated “very strong/very frequent”.

To evaluate nasal congestion symptoms, participants completed the Nasal Congestion Score (NCS), which employed a self-evaluation of congestion symptoms on a 4-point scale: 0 (absent symptoms), 1 (mild symptoms), 2 (moderate symptoms), and 3 (severe symptoms) [44]. The NCS allowed for the subjective assessment of nasal congestion severity experienced by each participant. Characteristics of the study populations are summarized in Table 1.

Table 1

Characteristics of the study populations.
Study populations		COVID-19	Olfaction
HT0	Hyposmic group (n = 36)	3–18 months before enrollment	Persistent post-acute olfactory dysfunction (TDI < 30)
HT1	Hyposmic group after olfactory training (n = 36)	3–18 months before enrollment	HT0 group, tested again after 3 months of olfactory training
CG1	1st control group (n = 18)	3–18 months before enrollment	Normosmia (TDI > 30); olfactory dysfunctions for less than 4 weeks duration after acute infection.
CG2	2nd control group (n = 18)	Never	Normosmia (TDI > 30)

HT0: hyposmic group before olfactory training; HT1: hyposmic group re-tested after 3 months of olfactory training; CG1: individuals with normal olfactory function (normosmia) who could have experienced olfactory dysfunctions for less than 4 weeks after acute COVID-19 infection; CG2: individuals with normosmia who have never experienced COVID-19 infection; TDI: Threshold, Discrimination, and Identification score obtained from the Sniffin' Sticks extended test n-Butanol, where TDI < 30 indicates olfactory defects, and TDI > 30 represents normosmia.

2.3. Olfactory rehabilitation and follow-up

After the initial assessment (HT0), each patient underwent a 12-week olfactory training program (HT1), which involved daily exposure to four intense odors: phenyl ethyl alcohol (rose), eucalyptol (eucalyptus), citronellal (lemon), and eugenol (cloves) [45, 46]During the olfactory training, each olfactory stimulus lasted for 10 consecutive seconds, using one substance at a time, with a 45-second break before the next odorant presentation. This routine was repeated three times, twice daily, resulting in a 10-minute complete session each time.

At the end of the 12-week follow-up, the participants underwent reassessment using the Sniffin’ Sticks extended test n-Butanol, nasal endoscopy, the brief QOD questionnaire, and the Nasal Congestion Score (NCS). Additionally, new samples of nasal brush and peripheral venous blood were collected for further analysis.

2.4. Inclusion and exclusion criteria

Inclusion criteria: SARS-CoV-2 infection diagnosed by positive nasopharyngeal swab 3–18 months before enrollment and persistent post-acute olfactory dysfunctions (TDI score < 30). Control groups subjects: SARS-CoV-2 infection diagnosed by positive nasopharyngeal swab during the 3–18 months preceding the recruitment and olfactory defects less than 4 weeks duration for the first group (CG1); no positive nasopharyngeal swab neither olfactory defect in the personal anamnesis of the second control group (CG2).

Exclusion criteria: suspicious or confirmed contact with positive case or attributable symptoms of SARS-CoV-2 infection in the 14 days prior enrollment; autoimmune disorders; primary or secondary immunodeficiency; use of immune system interacting and/or psychotropic drugs; positive history of head trauma, neurosurgery, sino-nasal surgery and neuropsychiatric or other sino-nasal diseases; occupational and/or voluptuous exposure to inhalants; olfactory deficits prior to SARS-CoV-2 infection.

2.5. Collection of nasal and blood samples

Under nasal endoscopic view, all participants underwent non-invasive olfactory mucosa brushing. A commercially available cytology brush (GIMA Brush, GIMA, Gessate, Italy) was placed at the level of the olfactory cleft and removed gently whilst being rotated three times. After sampling, the brush was shaken in an Eppendorf containing 1 mL phosphate buffered saline (PBS). The cell suspension was then loaded into a centrifuge 5430 R (Centrifuge, Eppendorf AG product, Hamburg, Germany) and spun for 5 min at 1500 r.p.m. The resulting supernatant (~ 850 µL) was then placed in plastic tubes and stored at -80° C.

Serum samples were collected in dry tubes by peripheral venous sampling, then stored in Eppendorf tubes at -80°C after centrifugation for 15 min at 4000x g.

2.6. Circulating calprotectin assay

The “QUANTA Flash Calprotectin'' kit (INOVA Diagnostics Inc., San Diego, CA, USA) was used for the quantitative calprotectin evaluation in nasal and blood samples. This chemiluminescent “sandwich” immunoassay was run at 37°C on the automated BIOFLASH Instrument (INOVA Diagnostics Inc., San Diego, CA, USA). Anti-calprotectin specific antibodies, coated on paramagnetic beads, were combined in a cuvette with patient sample and the assay buffer. The beads were then magnetized and washed several times. Isoluminol-conjugated calprotectin-specific capture antibodies were then added, generating a luminescent reaction when “trigger” reagents were mixed to the cuvette. The BIO-FLASH optical system measured the light obtained from this reaction as relative light units (RLUs). The resulting RLU values were proportional both to the amount of bound isoluminol conjugate and to the amount of captured calprotectin on the beads surface.

An instrument-specific working curve, created by running three calibrators, was used by the software to calculate ng/ml values from the RLUs. The calprotectin concentration values were multiplied for a k factor of 0.023 to convert in µg/ml, due to the 1:23 pre-dilution factor performed by the BIO-FLASH Instrument on the samples measured with this kit.

2.7 Statistical analysis

Results were calculated by Mann–Whitney test. Statistical significance was defined as p < 0.05. All data were analyzed using GraphPad Prism Software 9.1.1 (San Diego, California, USA).

3.1. Clinical evaluation and follow-up

All data on the hyposmic patients have been collected at the enrollment (HT0) and 3 months after olfactory training (HT1). As expected, the control groups CG1 and CG2 resulted normosmic (TDI 38 and 37.2; range 35–43 and 31–48, respectively) and the TDI score was low in HT0 patients (mean value 17,5; range 7–26), with 62% of patients resulting hyposmic and 38% anosmic at the Sniffin’ Sticks extended test. After three months of olfactory training, all HT1 patients resulted as normosmic (100%), with a mean TDI score of 34 (range 31–38).

These results were consistent with the improvement of the brief version of the Questionnaire of Olfactory Disorders (brief QOD). Nasal congestion was excluded by a low NCS score (mean value 0,6 in HT0 and HT1 patients, range 0–1), while concomitant nasal pathologies were excluded by the Lund Kennedy endoscopic score. Clinical results are summarized in Table 2.

Table 2

Clinical characteristics and Olfactory Parameters of the study groups
		Age (years)	TDI	QOD-VAS	QOD-P + QOD-QOL	NCS
HT0	Mean Range	53 (24–72)	17,5 (7–26)	23,85 (3–30)	21 (0–28)	0,6 (0–1)
HT1	Mean Range	53 (24–72)	34,0 (31–38)	12,3 (0–16)	8 (0–15)	0,5 (0–1)
CG1	Mean Range	46 (23–68)	38,0 (35–43)	0	0	0,6
CG2	Mean Range	45 (23–70)	37,2 (31–48)	0	0	0,6

HT0: Hyposmic group before olfactory training; HT1: Hyposmic group after 3 months of olfactory training; CG1: 1st control group; CG2: 2nd Control Group; TDI: Threshold, Discrimination, and Identification score for olfactory function; QOD-VAS: Questionnaire of Olfactory Disorders Visual Analogue Scale; QOD-P: Questionnaire of Olfactory Disorders specific to parosmia; QOD-L: Questionnaire of Olfactory Disorders specific to quality of life; NCS: Nasal Congestion Score.

3.2. Local and serum circulating calprotectin

Circulating calprotectin levels were analyzed in serum and in nasal supernatant of the hyposmic patients at the enrollment (HT0) and 3 months after olfactory training (HT1), in comparison to the control groups: 18 subjects recovered from COVID-19 without hyposmia (CG1) and 18 healthy controls never infected by SARS-CoV-2 (CG2). Results are shown in Table 3, Fig. 1.

In supernatant samples, the median cCLP concentrations were statistically significant higher (p < 0,05) in Hyposmic group (HT0, 10.90 µg/ml, range 3.70-14.31 µg/ml) respect to the two control groups (CG1 and CG2), where the values were similar (3.83 vs. 3.92 µg/ml; range 1.94–7.3 µg/ml and 2.94–4.82 µg/ml, respectively). In hyposmic patients (HT1) after three months of olfactory training, a strong decrease of cCLP concentration was detected (1.97 µg/ml, range 1.46–4.32 µg/ml), thus representing the lower value when compared both to HT0 and to the two control groups.

Regarding the median cCLP concentrations in serum samples, they showed a similar trend to nasal supernatant: higher values were obtained in the Hyposmic T0 group (8.32 µg/ml, range 6.77–10.09 µg/ml), with progressively lower values in the two control groups CG1 and CG2 (6.82 µg/ml and 7.35 µg/ml; range 5.67–7.28 µg/ml and 5.35–8.29 µg/ml, respectively) and in the Hyposmic group after OT (5.38 µg/ml, range 3.81–7.45 µg/ml). Data were statistically significant with a p-value < 0,005 between Hyposmic T0 vs. Hyposmic T1, and a p-value < 0,05 between Hyposmic T0 and CG1.

Table 3

Median, range and SD values of circulating calprotectin (cCLP) values in blood samples and nasal supernatant in the four study populations.
Study population		cCLP in blood samples	cCLP in nasal supernatant
HT0	Median (µg/ml) (range)	8.32 µg/ml (6.77–10.09)	10.90 µg/ml (3.70–14.31)
HT0	SD	± 2.06	± 8.20
HT1	Median (µg/ml) (range)	5.38 µg/ml (3.81–7.45)	1.97 µg/ml (1.46–4.32)
HT1	SD	± 2.02	± 4.64
CG1	Median (µg/ml) (range)	6.82 µg/ml (5.67–7.28)	3.83 µg/ml (1.94–7.30)
CG1	SD	± 1.02	± 2.50
CG2	Median (µg/ml) (range)	7.35 µg/ml (5.35–8.29)	3.92 µg/ml (2.94–4.82)
CG2	SD	± 1.83	± 2.00

cCLP: circulating calprotectin; HT0: Hyposmic group before olfactory training; HT1: Hyposmic group after 3 months of olfactory training; CG1: 1st Control Group; CG2: 2nd Control Group.

The pathophysiology of persistent olfactory impairments in post-COVID-19 patients remains enigmatic, with a complex interplay of various factors contributing to their development. Geographical, ethnic, genetic, and host-specific variations might all play a role in determining susceptibility. Nonetheless, the specific fundamental mechanism that distinguishes acute infection from post-acute sequelae of COVID-19 remains to be elucidated.

One plausible explanation lies in the establishment of a persistent nasal inflammatory micro-environment subsequent to the primary infection. This inflammatory milieu can lead to changes in mucus volume and composition, rendering it challenging for odorants to effectively bind to olfactory receptors, thereby impairing the sense of smell[47]. Additionally, olfactory defects in post-acute sequelae of COVID-19 have been associated with morphological changes observed in the olfactory bulbs and cortex, indicating potential neurodegenerative processes taking place [38, 39]. However, the causality and the temporal relationship between these changes and peripheral inputs due to epithelial damage warrant further investigation. Remarkably, ACE2 receptors, the primary binding sites for SARS-CoV-2 entry into cells, are found in higher concentrations on sustentacular cells than on olfactory neurons, which might explain why olfactory neurons are comparatively preserved [48].

To address the long-lasting olfactory dysfunctions, olfactory training (OT) has been recommended; it has demonstrated promising results in improving odor sensitivity and facilitating the reorganization of olfactory neural networks, contributing to the restoration of olfactory function [49]. Notably, the presence of ciliary neurotrophic factor, produced by olfactory ensheathing cells, might enhance olfactory receptor responsiveness to OT through its effects on mRNA levels and protein expression, further supporting the benefits of olfactory rehabilitation[50]. Olfactory training is an extremely easy and safe therapy without significant side effects, particularly beneficial when started within 12 months from the onset of the olfactory disorder.

Our results shed light on the potential role of calprotectin as a promising biomarker in post-acute sequelae of COVID-19 and olfactory dysfunctions showing significantly higher levels of circulating calprotectin in patients with PASC persistent olfactory dysfunction (HT0) when compared to a healthy control group (CG2) and to subjects with previous COVID-19 without olfactory sequelae (CG1), both in serum and nasal supernatant samples, where it was particularly visible. Furthermore, finding increased levels of calprotectin in nasal samples of patients with long-lasting olfactory defects is a surprising yet intriguing evidence, since it allows to indicate the persistence of a local post-acute inflammatory status as a main factor in the establishment of an enduring olfaction disease. The comparison of calprotectin levels before (HT0) and after the restoration of the olfactory function througholfactory training (HT1) showed that all patients presented a significant cCLP decrease in concomitance to the improvement of the olfactory function, as evidenced by the TDI Sniffin’ Test extended and by the Questionnaire of Olfactory Disorders, providing valuable insights into the potential of olfactory rehabilitation strategies. The decrease in calprotectin levels in serum samples and, in particular, in nasal supernatant samples highlighted the role of the circulating calprotectin as well as of the olfactory training protocols not only in the treatment of this condition but also in the modulation of abnormal pro-inflammatory responses, possibly contributing to the restoration of olfactory function.

The main limitation of our study is the small number of participants. However, due to the rarity of the condition, patients are often misdiagnosed or did not come to medical attention and infrequently accept to pursue three months of olfactory training. For these reasons, our results can offer an insight into useful information also for this study population, given the low adherence to the olfactory rehabilitation protocol.

Several studies have proposed the circulating calprotectin function as a monitoring tool for various inflammation-related diseases, as well as a biomarker of subclinical inflammation in patients. To the best of our knowledge, this is the first study to investigate the role of calprotectin in post-acute sequelae of COVID-19 presenting with long-lasting olfactory defects. The observed increase in calprotectin levels in nasal and its subsequent decrease with olfactory training could suggest the utility of calprotectin as a marker of inflammation in these conditions. Moreover, our results give new insights to recent literature, proposing circulating calprotectin as a new, quantitative and convenient marker in the assessment and the monitoring of nasal diseases. Nevertheless, further studies are required to correlate biochemical results to nasal cytology, in order to shed light on the mechanisms underlying these findings.

In conclusion, our data have the potential to enhance the understanding of the role of circulating CLP in olfactory impairments and may provide new avenues for improved management and treatment strategies in affected individuals. Additionally, it may offer valuable information about the broader application of cCLP as a biomarker in various inflammation-driven diseases and contribute to refining risk stratification strategies.

Author contributions:

Beatrice Francavilla, Denise Fiorelli, Gianluca Velletrani and Marzia Nuccetelli contributed to the study conception and design. Material preparation, data collection and analysis were performed by Beatrice Francavilla, Denise Fiorelli, Gianluca Velletrani and Sara Maurantonio. The first draft of the manuscript was written by Beatrice Francavilla, Denise Fiorelli, Gianluca Velletrani and Francesco Maria Passali, Sergio Bernardini, Stefano Di Girolamo and Marzia Nuccetelli commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Funding

No funding was received for conducting this study.

Conflict of interest

The authors have no relevant financial or non-financial interests to disclose.

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Circulating Calprotectin as a Potential Biomarker of Persistent Olfactory Dysfunctions in Post-COVID-19 Patients

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Journal Publication

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Abstract

Objective and Design:

Materials and Methods

Results

Conclusions

Figures

1. Introduction

2. Materials and methods

2.1. Study design and population

2.2. Clinical evaluation

2.3. Olfactory rehabilitation and follow-up

2.4. Inclusion and exclusion criteria

2.5. Collection of nasal and blood samples

2.6. Circulating calprotectin assay

2.7 Statistical analysis

3. Results

3.1. Clinical evaluation and follow-up

3.2. Local and serum circulating calprotectin

4. Discussion

Declarations

References

Additional Declarations

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Journal Publication

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