key: cord-0875936-iw239le6
authors: Maniscalco, Mauro; Ambrosino, Pasquale; Poto, Remo; Fuschillo, Salvatore; Poto, Sergio; Matera, Maria Gabriella; Cazzola, Mario
title: Can FeNO be a biomarker in the post-COVID-19 patients monitoring?
date: 2022-01-26
journal: Respir Med
DOI: 10.1016/j.rmed.2022.106745
sha: c8113bc2f781ab3f0feaa727711f76898e2beca3
doc_id: 875936
cord_uid: iw239le6

The nature of the inflammatory and fibrotic processes found in patients with post-COVID-19 syndrome makes it possible to speculate that in such patients fractional exhaled nitric oxide (FeNO) may be a useful biomarker. Consequently, we set out to verify the consistency of this hypothesis. We consecutively enrolled 68 post-COVID patients after being hospitalized for persistent clinical manifestations within 2 months from disease onset and 29 healthy volunteers as control group. None of post-COVID patients had bronchial asthma or were being treated with a corticosteroid. Only 19 out of 68 post-COVID-19 patients reported a FeNO value > 25 ppb. The mean FeNO value in post-COVID-19 patients was 18.55 ppb (95% CI: 15.50 to 21.58), while in healthy subjects it was 17.46 ppb (95% CI: 15.75 to 19.17). The mean difference was not statistically significant (P = 0.053). However, the mean FeNO value of post-COVID-19 patients was higher in men than in women (20.97 ppb; 95% CI: 16.61 to 25.33 vs 14.36 ppb; 95% CI: 11.11 to 17.61) with a difference between the two sexes that was statistically significant (P = 0.016). Mean FeNO was 14.89 ppb (95% CI: 10.90 to 18.89) in patients who had been treated with systemic corticosteroids because of their COVID-19, and 20.80 ppb (95% CI: 16.56 to 25.04) in those who had not taken them, with a difference that was statistically significant (P = 0.043). The data generated in this study suggest that measurement of FeNO is not useful as a biomarker in post-COVID-19 patient. However, this hypothesis needs solid validation with additional specifically designed studies.

A high proportion of patients recovering from COVID-19 reported persistence of symptoms, particularly fatigue and dyspnea, even 2 months after being discharged [1] .

This delayed recovery of symptoms has been termed "post-COVID-19 syndrome" or "long COVID" [2] . The pathogenesis of post-COVID syndrome remains largely unknown.

Evidence suggests that prolonged inflammation, nervous system dysfunction, endothelial damage, and thromboembolism have a key role in the pathogenesis of most post-COVID manifestations [3] . Furthermore, there is increasing evidence that fibrotic changes and interstitial lung abnormalities may result from COVID-19 infection in some cases, although we still do not know whether the fibrosis is stable or progressive [4] .

The high proportion of patients who develop the post-COVID-19 syndrome is the reason for the frenetic search for biological markers that are clinically useful in predicting a severe disease course in COVID-19 patients and also responses to treatment [5] .

Although not yet widely implemented, exhaled nitric oxide (FeNO) fraction has emerged in recent years as a potentially useful biomarker for the assessment of airway inflammation in both undiagnosed patients with nonspecific respiratory symptoms and in those with established airway disease [6] . FeNO is widely accepted as a non-invasive biomarker of inflammation and oxidative stress in the lungs [7] . There are also reports of increased FeNO fraction values in interstitial lung disease with alveolar NO (CaNO) concentrations that correlate with 6-minute walking distance, oxygen saturation recovery time, total lung capacity, and forced vital capacity (FVC) [8] .

The nature of the inflammatory and fibrotic processes found in patients with post-COVID-19 syndrome makes it possible to speculate that in such patients FeNO may be a useful biomarker. However, the impact of respiratory viruses on FeNO levels has not yet been clarified.

In children with an acute asthma exacerbation, FeNO levels rise to a greater extent in those whose exacerbation is not virus-induced [9] . In contrast, another study has observed an increase in FeNO levels following human rhinovirus 16 infection in asthmatics [10] . In addition, respiratory syncytial virus (RSV) has been shown to induce inducible NO synthase (iNOS)-mediated expression of Kruppel-like transcription factor 6 in human alveolar epithelial type 2 cells [11] .

These observations suggest that FeNO and CaNO can be considered to be markers of epithelial damage in the proximal and distal airways, respectively, during viral infections e.g. SARS-CoV-2 [12] .

In any case, upregulation of iNOS, which leads to an increase in the concentration of NO, is induced by pro-inflammatory cytokines that are highly upregulated in patients with COVID-19. It has been shown that in SARS-CoV-2 infection, tumour necrosis factor α and interferon γ synergistically induce iNOS and NO, which subsequently induce cell death [13] .

Considering that the prolonged inflammation that characterises post-COVID-19 syndrome and its possible impact on NO production, we aimed to test whether FeNO, which has the advantage of being standardized, quick, non-invasive, simple, and easy to reproduce, could be considered a valid biomarker in patients with post-COVID-19 syndrome.

In this prospective study, convalescent (post-) COVID-19 patients were consecutively selected for entry into the study after being admitted within less than 2 months after the onset of COVID-19 to the Istituti Clinici Scientifici Maugeri IRCCS, Telese Terme, Benevento, Italy, or the Ospedale Universitario "San Giovanni di Dio e Ruggi d'Aragona", J o u r n a l P r e -p r o o f Salerno, Italy, to undergo a pulmonary rehabilitation program or to continue appropriate medical care. All patients had two consecutive negative SARS-CoV-2 swab tests before their admission in our wards. An additional control group of 29 healthy volunteers enrolled among hospital staff from January 2021 to April 2021 was also included in the analysis. All volunteers tested negative for SARS-CoV-2 at study entry and had no history of positive nasopharyngeal swab at periodic monitoring of hospital staff.

Exclusion criteria for cases and controls were: age < 18 years; history of atopy and/or asthma; previously documented eosinophilic airway inflammation; peripheral blood eosinophil count at admission > 500/μL (> 0.5 × 10 9 /L); current smokers or ex-smokers; history of chronic obstructive pulmonary disease; history of bronchial asthma; any other respiratory condition other than COVID-19 for case; history of cardio-or cerebrovascular events; any condition associated with poor compliance with the study protocol or inadequate understanding of the study procedures; being treated with systemic or inhaled corticosteroids.

The study, which was reported following the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) recommendations to limit known sources of bias [14] , was conducted in accordance with the 1975 Declaration of Helsinki of the World Medical Association. The Institutional Review Board of Istituto Nazionale Tumori, Fondazione Pascale, Naples, Italy approved this study with reference number ICS11/20. All patients provided written informed consent to use their de-identified data.

After informed consent signature, the main demographic and clinical information pertaining to the acute phase of COVID-19, pulmonary function, and ongoing treatments were collected for all post-COVID-19 patients. For control subjects, persistently anonymized data were analyzed after collection. FeNO measurements were performed in all participants according to ATS/ERS guidelines [16] . Briefly, all evaluations were performed at a standardized exhalation flow rate of 50 ml/s. To perform a valid exhalation maneuver, the flow parameters were controlled by both audio and visual feedback supplied by manufacturers, allowing the participant to maintain a constant exhaled breath flow rate. FeNO measurements were obtained in duplicate.

Subjects were instructed to avoid exercise and consumption of nitrate-rich diets at least 2 hours before measurement.

Statistical analysis was performed using Prism 8 software package (GraphPad Software Inc, USA). Data were expressed as mean and 95% confidence interval (CI) (standard errors have been reported in figures). Comparison between healthy subjects and convalescent COVID-19 patients was made with unpaired t-test and Welch's correction.

Relationships between continuous variables were examined using simple regressions with J o u r n a l P r e -p r o o f Pearson's correlation coefficient (r). All results were expressed as 2-tailed values, P values < 0.05 being statistically significant.

Sixty-eight post-COVID patients were admitted into the study. The control group included 29 healthy volunteers. The main demographic and clinical characteristics of the study participants have been reported in Table 1 . Functional parameters (FEV1% predicted and FVC% predicted) showed statistically lower mean values in post-COVID patients compared with those in the control group (table 1) . It must be noted that the spirometric data refer to 62 patients because other 6 subjects were unable to perform the pulmonary function test when they were admitted to our wards. 

The data generated in this study suggest that measurement of FeNO is not useful as a biomarker of post-COVID-19 patient monitoring. This conclusion correlates well with the J o u r n a l P r e -p r o o f observation that eosinophils do not play a protective or exacerbating role during SARS-CoV-2 infection [19] . In addition, it is eosinopenia that is very common and often pronounced in cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection [20] . [21] . However, while in our population we found no predominant pulmonary fibrotic changes on radiological investigation, in Cameli's group such changes and ground glass opacities were associated with high CaNO levels although no further significant differences were observed between other FeNO parameters and CT scan data.

In a small Finish study, mean FeNO value was 21.7 ppb three to six months after hospital discharge [22] . Seven out of 20 patients had slightly elevated FeNO of 25 to 50 ppb, and none of the participants had an abnormal FeNO of more than 50 ppb.

In a study conducted in Saudi Arabia, the median FeNO value measured in 20 patients at least 3 months post-recovery was 19 ppb, while in a control group of 30 individuals it was 16 ppb [23] .

Our data do not allow us to understand whether FeNO values are elevated in patients with COVID-19, with possible differences between those with versus without pneumonia or acute respiratory distress syndrome, and then decrease in the post-COVID phase.

The documentation that FeNO mean values in post-COVID patients were quite similar to those in the control group suggests that COVID-19 does not induce chronic bronchial inflammation or predispose to chronic obstructive diseases. This hypothesis, which needs solid validation with additional specifically designed studies, contrasts with the evidence of persistent inflammation 5 months after the onset of olfactory symptoms in patients with COVID-19 who recovered from their olfactory dysfunction compared with patients who did J o u r n a l P r e -p r o o f not experience anosmia [24] . However, in the same study it was also demonstrated the presence of comparable FeNO and CaNO between COVID-19 patients with and without olfactory dysfunction. This suggests that the increase in residual inflammation accompanying olfactory loss is limited only to the olfactory epithelium probably due to the increased expression of both angiotensin-converting enzyme 2 and angiotensin-converting enzyme 2 in the ciliated and goblet cells of the nasal cavity compared with the lower airway and alveolar epithelial cells. It has been suggested to measure nasal NO levels in patients with post-acute COVID-19 syndrome with persistent anosmia to evaluate ongoing background activation of iNOS by proinflammatory cytokines even after recovery [24] . This is an interesting possibility that could provide additional information about the level of airway inflammation [25] . However, its validity needs to be appropriately demonstrated.

The evidence that FeNO was significantly higher in males than in females corresponds to the accepted notion that sex is an important factor determining FeNO measurements [26] , but not necessarily because of lower NO production in women but because women have smaller lungs and consequently higher linear flow velocities in the airways, and it is known that as FeNO is highly flow-dependent, it decreases with increasing flow [27] . This suggests that the significant difference we recorded between the sexes does not appear to be a consequence of suffering from COVID-19.

The documentation that FEV1% and FVC% predicted mean values were statistically lower in post-COVID-19 patients compared with those in the control group confirms that COVID-19 pneumonia can cause significant alterations in lung function [28] . Our patients were examined within 8 weeks after the onset of COVID-19, and it was reported that 54% of COVID-19 survivors had abnormal lung function 10 weeks after diagnosis [29] .

The fact that patients treated in the acute phase with corticosteroids had lower mean FeNO values than those not treated with such agents is another expected result. While we J o u r n a l P r e -p r o o f have no information on whether the use of corticosteroids was in accordance with the recommendations available for hospitalised patients with acute COVID-19 [30] because such treatment was administered in different hospital settings than in our facilities, it is known that treatment with systemic corticosteroids induces a drastic reduction in FeNO values, although in non-asthmatic subjects it is difficult to predict whether they rise again and, if so, after how long [31] . In any case, a small study found FeNO levels within the normal range during acute symptoms of SARS-CoV-2 infection, with some increase during the recovery phase, independently of disease severity or the patient's history of atopy.

However, in patients treated with corticosteroids, a significant FeNO decrease during clinical evolution was more pronounced [32] .

Overall, the preliminary data generated in this study suggest that measurement of FeNO is not useful as a biomarker in post-COVID-19 patient monitoring. A major limitation of this pilot study is the small sample, thus suggesting the need for larger studies specifically designed to test the usefulness of this biomarker in post-acute care and rehabilitation settings. J o u r n a l P r e -p r o o f

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The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 

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