*Article* **Study of Nasal Fractional Exhaled Nitric Oxide (FENO) in Children with Allergic Rhinitis**

**Sy Duong-Quy 1,2,3,\* ,†, Thuy Nguyen-Thi-Dieu 4,†, Khai Tran-Quang <sup>5</sup> , Tram Tang-Thi-Thao <sup>1</sup> , Toi Nguyen-Van <sup>1</sup> , Thu Vo-Pham-Minh <sup>5</sup> , Quan Vu-Tran-Thien <sup>6</sup> , Khue Bui-Diem <sup>6</sup> , Vinh Nguyen-Nhu <sup>6</sup> , Lam Hoang-Thi <sup>7</sup> and Timothy Craig <sup>3</sup>**


**Abstract:** (1) Background: Exhaled nitric oxide (NO) has been considered as a biomarker of airway inflammation. The measurement of fractional exhaled NO (FENO) is a valuable test for assessing local inflammation in subjects with allergic rhinitis (AR). (2) Objective: To evaluate (a) the correlation between nasal FENO with anthropometric characteristics, symptoms of AR and nasal peak flows in children without and with AR; and (b) the cut-off of nasal FENO for diagnosis of AR in symptomatic children. (3) Methods: The study was a descriptive and cross-sectional study in subjects with and without AR < 18 years old. All clinical and functional characteristics of the study subjects were recorded for analysis. They were divided into healthy subjects for the control group and subjects with AR who met all inclusion criteria. (4) Results: 100 subjects (14 ± 3 years) were included, including 32 control subjects and 68 patients with AR. Nasal FENO in AR patients was significantly higher than in control subjects: 985 ± 232 ppb vs. 229 ± 65 ppb (*p* < 0.001). In control subjects, nasal FENO was not correlated with anthropometric characteristics and nasal inspiratory or expiratory peak flows (IPF or EPF) (*p* > 0.05). There was a correlation between nasal FENO and AR symptoms in AR patients and nasal IPF and EPF (*p* = 0.001 and 0.0001, respectively). The cut-off of nasal FENO for positive AR diagnosis with the highest specificity and sensitivity was ≥794 ppb (96.7% and 92.6%, respectively). (5) Conclusion: The use of nasal FENO as a biomarker of AR provides a useful tool and additional armamentarium in the management of allergic rhinitis.

**Keywords:** nitric oxide; NO; exhaled NO; FENO; allergic rhinitis; nasal peak flow

#### **1. Introduction**

In the upper airway, exhaled nitric oxide (NO) is produced mainly from the rhinosinusitis mucosa. It can be measured by non-invasive techniques using devices with chemiluminescence or electroluminescence methods [1–3]. The main source of nasal NO is consistently generated from the nasal mucosa and perinasal sinus epithelium, where inducible nitric oxide synthase (iNOS) is present. In the upper airway, the role of nasal NO has been described as regulating airway function, providing non-specific protection against

**Citation:** Duong-Quy, S.;

Nguyen-Thi-Dieu, T.; Tran-Quang, K.; Tang-Thi-Thao, T.; Nguyen-Van, T.; Vo-Pham-Minh, T.; Vu-Tran-Thien, Q.; Bui-Diem, K.; Nguyen-Nhu, V.; Hoang-Thi, L.; et al. Study of Nasal Fractional Exhaled Nitric Oxide (FENO) in Children with Allergic Rhinitis. *Sinusitis* **2021**, *5*, 123–131. https://doi.org/10.3390/ sinusitis5020013

Academic Editor: Eleonora Nucera

Received: 9 August 2021 Accepted: 3 October 2021 Published: 8 October 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

infection related to its destructive property. Nasal NO also contributes to upper airway protection due to its role in regulating ciliary motility, and low nasal NO levels are usually associated with decreased upper airway ciliary function [4]. Nasal NO has been proposed in the hypothesis of humidifying and warming of inhaled air through the nasal passage.

The alteration in nasal fractional exhaled NO (nasal FENO) levels has been described previously in various diseases such as allergic rhinitis (AR), primary ciliary dyskinesia (PCD), cystic fibrosis, and sinusitis [5–8]. Therefore, the measure of nasal FENO is now considered a useful biomarker in clinical practice for patients with rhino-sinusitis diseases. In patients with PCD, nasal FENO measurement is routinely performed for screening this genetic disorder [9]. In patients with AR, nasal FENO has been used to manage the disease in the same manner as FENO in patients with asthma [10]. The increased iNOS expression and activity due to contact with airborne allergens induces the production of nasal FENO in patients with AR. While the correlation between exhaled NO and lower airway inflammation in asthmatic patients due to eosinophils has been demonstrated, the application of nasal FENO measurement in patients with AR is relatively complex and remains controversial.

Hence, this study was conducted to evaluate (1) the correlation between nasal FENO and anthropometric characteristics, symptoms of AR, and nasal peak flows in children without and with AR; and (2) the cut-off of nasal FENO for diagnosis of AR in symptomatic children.

#### **2. Methods**

#### *2.1. Patients*

Patients with a diagnosis of AR were included in the current study when they were referred to the Clinical Research Center of Lam Dong Medical College for measuring nasal FENO and a skin-prick test. The present study was approved by the IRB of Lam Dong Medical College, Dalat, Vietnam (ID: CDYTLD.NCKH.03.2018); signed written informed consent was obtained from all the study subjects. The study followed the principles of the 1964 Declaration of Helsinki.

#### 2.1.1. Inclusion Criteria

Patients <18 years old with AR symptoms (nasal congestion, runny nose, nasal itching, or sneezing) lasting more than 4 days per week and for more than 4 consecutive weeks were classified into the AR group.

#### 2.1.2. Exclusion Criteria

The exclusion criteria were one of the following features: severe cardiorespiratory disease, AR treated with oral or local corticosteroids, septal deviation or nasal polyp diagnosed, and upper or lower airway infection in the past 15 days; subjects unable to undergo the functional laboratory testing were also excluded from the present study.

#### *2.2. Methods*

This was a cross-sectional and descriptive study. All clinical and functional parameters were recorded for analysis. Included study subjects were divided into 2 groups: a control group consisting of healthy people without nasal and sinus diseases, and the AR group consisting of patients who met the selection criteria.

The criteria for a diagnosis of AR were: having one of the symptoms of nasal congestion, nasal itching and sneezing, and a runny nose lasting more than 4 days/week according to the season or occurring when exposed to respiratory allergens (dog or cat fur, pollen, mold, and house dust mites) in the living or working environment [11].

#### 2.2.1. Laboratory Functional Testing

The peak inspiratory and expiratory flows (PIF and PEF) in the nose were measured by using a nasal mask-attached peak-flow meter device (Mediflux, Bry Sur Marne, France). Nasal FENO measurement was performed by using multi-flow exhaled NO (Hypair NO,

Medisoft; B-5503 Sorinnes; Belgium). Nasal FENO measurement was carried out according to the manufacturer's instructions.

#### 2.2.2. Statistical Analyses

SPSS 22.0 software (Chicago, IL, USA) was used to analyze all the collected data. Categorical variables are presented as numbers or percentages. Continuous parameters are presented as means ± standard deviation (SD). The skewness–kurtosis test measured the normal distribution. The Mann–Whitney U test was used for the comparison of means between groups. The correlation between nasal FENO and quantitative variables with normal distribution was examined by regression analysis. A *p*-value < 0.05 was considered statistically significant.

#### **3. Results**

From January 2018 to December 2019, 100 subjects participated in the study, including 32 healthy people (control group) and 68 patients diagnosed with AR (AR group). The latter met the selection criteria and performed all the required functional tests.

#### *3.1. Clinical and Functional Characteristics Study Subjects*

There was no significant difference between the AR group and control group regarding age, gender, height, weight, and BMI (*p* > 0.05; Table 1). The proportion of AR patients who had symptoms of blocked nose, nasal itching or sneezing, and runny nose was 97%, 100%, and 100%, respectively (Table 1). Peak inspiratory and expiratory volumes in patients with AR were significantly lower than in the control group (*p* < 0.01 and *p* < 0.01; Table 1). The mean nasal FENO was considerably higher in the AR group than in the control group (985 ± 232 ppb vs. 229 ± 155 ppb; *p* < 0.001; Table 1).


**Table 1.** Clinical and functional characteristics of study subjects.

*p* \*: different between AR group and control group; AR: allergic rhinitis; BMI: body mass index; FENO: fractional exhaled nitric oxide; L: liter; ppb: parts per billion; NA: not applicable.

#### *3.2. Correlation between Nasal FENO and the Anthropometric Characteristics of the Control Subjects and Clinical Symptoms in Patients with AR*

There was no significant correlation between nasal FENO and the anthropometric characteristics of the control subjects participating in the present study (N = 32; Table 2). Nasal FENO had a significant mild to moderate correlation with clinical symptoms of AR, including blocked nose, itching or sneezing, and runny nose (R = 0.356, 0.679 and 0.587; *p* < 0.001, 0.0001 and 0.001, respectively; N = 68; Table 2). *Sinusitis* **2021**, *5*, x 4 of 8 Nasal FENO, ppb618 ± 395 (124–1385) 985 ± 232 (526–1385) 229 ± 65 (152–299) <0.001

**Table 2.** Correlation between nasal FENO and the anthropometric characteristics of the control subjects and with clinical symptoms in patients with AR. *p* \*: different between AR group and control group; AR: allergic rhinitis; BMI: body mass index; FENO: fractional exhaled nitric oxide; L: liter; ppb: parts per billion; NA: not applicable.


AR: allergic rhinitis; BMI: body mass index; FENO: fractional exhaled nitric oxide.

#### *3.3. Correlation between Nasal FENO and Nasal Peak Flow of Study Subjects* **Table 2.** Correlation between nasal FENO and the anthropometric characteristics of the control subjects and with clinical symptoms in patients with AR.

There was no significant correlation between nasal FENO and inspiratory and expiratory peak flow in subjects without AR (control subjects; Table 3). There was a significant and negative linear correlation between nasal FENO and peak inspiratory flow (R = −0.462; *p* = 0.0012; Table 3, Figure 1a) and peak expiratory flow (R = −0.378; *p* = 0.0016; Table 3, Figure 1b). **Correlation Anthropometric Parameters (Control Subjects; N = 32) Symptoms of AR (AR Patients; N = 68) Nasal FENO Age Sex Height Weight BMI Blocked Nose Itching or Sneezing Runny Nose**  R 0.098 0.325 0.094 0.082 0.076 0.356 0.679 0.587



AR: allergic rhinitis; FENO: fractional exhaled nitric oxide. Table 3, Figure 1b).

**Figure 1.** (**a**) Correlation between nasal FENO and peak inspiratory flow in patients with AR. (**b**) Correlation between nasal FENO and peak expiratory flow in patients with AR. AR: allergic rhinitis; FENO: fractional exhaled nitric oxide. **Figure 1.** (**a**) Correlation between nasal FENO and peak inspiratory flow in patients with AR. (**b**) Correlation between nasal FENO and peak expiratory flow in patients with AR. AR: allergic rhinitis; FENO: fractional exhaled nitric oxide.

**Peak Expiratory Flow** 

R 0.095 0.074 −0.462 −0.378

**AR Patients (N = 68)** 

> **Peak Expiratory Flow**

**Peak Inspiratory Flow** 

**Flow** 

**Correlation Control Subjects** 

**Table 3.** Correlation between nasal FENO and nasal peak flow of study subjects.

#### *3.4. Cut-Off of Nasal FENO in the Diagnosis of AR in Children* the highest Youden index was equivalent to the most significant area under the ROC

The cut-off of nasal FENO in positive diagnoses of AR is presented in Figure 2 and Table 4 (N = 100). The results of ROC curve analysis showed that the cut-off of FENO with the highest Youden index was equivalent to the most significant area under the ROC curve of 794 ppb and had a specificity and sensitivity of 96.7% and 92.6%, respectively. (Figure 2, Table 4). curve of 794 ppb and had a specificity and sensitivity of 96.7% and 92.6%, respectively. (Figure 2, Table 4).

AR: allergic rhinitis; FENO: fractional exhaled nitric oxide.

*3.4. Cut-Off of Nasal FENO in the Diagnosis of AR in Children* 

*Sinusitis* **2021**, *5*, x 5 of 8

P 0.324 0.417 0.0012 0.0016

The cut-off of nasal FENO in positive diagnoses of AR is presented in Figure 2 and

760 92.4 93.1 186.767

863 91.7 95.6 188.673

899 91.4 95.6 188.348

905 90.2 95.6 187.560

916 89.8 95.6 186.134

938 88.2 95.6 185.778

945 88.1 95.6 184.657

The results of our study demonstrated that: (1) Nasal FENO did not depend on

In healthy people, FENO concentrations in the nose are often much higher than in

anthropometric characteristics or nasal peak inspiratory or expiratory flows in children

without AR; (2) there was a correlation between nasal FENO and clinical symptoms, nasal

peak inspiratory, and expiratory flows in children with AR; and (3) the cut-off nasal FENO

the lower respiratory tract (300–800 ppb vs. 5–25 ppb). In the rhino-sinusal area, the

for a diagnosis of AR with the highest specificity and sensitivity was ≥794 ppb.

**(%) Youden Index** 

Table 4 (N = 100). The results of ROC curve analysis showed that the cut-off of FENO with

**Figure 2.** ROC curve of the nasal FENO cut-off for the diagnosis of AR. AR: allergic rhinitis; FENO: **Figure 2.** ROC curve of the nasal FENO cut-off for the diagnosis of AR. AR: allergic rhinitis; FENO: fractional exhaled nitric oxide.

fractional exhaled nitric oxide. **Table 4.** Cut-off nasal FENO with corresponding AR diagnosis sensitivity and specificity.


**4. Discussion** 

#### **4. Discussion**

The results of our study demonstrated that: (1) Nasal FENO did not depend on anthropometric characteristics or nasal peak inspiratory or expiratory flows in children without AR; (2) there was a correlation between nasal FENO and clinical symptoms, nasal peak inspiratory, and expiratory flows in children with AR; and (3) the cut-off nasal FENO for a diagnosis of AR with the highest specificity and sensitivity was ≥794 ppb.

In healthy people, FENO concentrations in the nose are often much higher than in the lower respiratory tract (300–800 ppb vs. 5–25 ppb). In the rhino-sinusal area, the paranasal sinuses are a vital source of nasal FENO production. Previously, Lundberg et al. [8] described that after perforation of the maxillary sinus, the continuous synthesis of NO at a very high concentration was detected. However, Hood et al. [12] showed that only NO concentrations measured in the nasal cavity came from the sinuses by diffusion due to the NO concentration difference between the nose and sinuses, but it was also produced in the nasal cavity. In the present study, the level of nasal FENO in children without AR symptoms was varied from 152 to 298 ppb (Table 1). This result is also consistent with the manufacturer's recommendation that the expected value of nasal FENO in children is less than 300 ppb.

The present study showed that, in control children, the level of nasal FENO was not correlated with anthropometric characteristics such as age, gender, height, weight, and BMI (Table 2). Thus, this is a prominent advantage of nasal FENO as a biomarker because it can be used to diagnose various pathological conditions of the nose regardless of demographic features. It might also be similar to bronchial FENO because a previous study also showed that bronchial FENO had no significant correlation with demographic characteristics [13]. However, the recommended cut-off of the normal value of nasal FENO has been established based on a large population that is representative and takes the age into account [14]. The present study only used a control group with a small sample size to determine the nasal FENO value in healthy children compared with AR children.

Because of the short half-life of NO in gas form, indirect methods were previously used to measure the NO concentration in the body during the humoral phase, based on the measurement of NO metabolism products such as nitrate and nitrite, or using immunohistochemistry techniques to determine NOS activity. In contrast to NO produced in tissue or the blood, exhaled NO in the airways is more stable, allowing us to measure it directly [15–17]. Various techniques have been used to measure exhaled NO concentration, and the most commonly used is the chemiluminescence method. This method is highly sensitive, and exhaled NO can be detected at levels as low as parts per trillion. A new NO analysis method based on the electroluminescence technique has been developed and used in clinical practice (Figure 3) [3,18]. This technique has been shown to have high accuracy and good correlation with other methods, and has the advantage of being small compared with fixed routine chemiluminescence analyzers.

The present study results showed that nasal FENO in children with AR was significantly higher than in children without AR (Table 1). Especially in patients with AR, there was a significant correlation between nasal FENO and clinical symptoms (Table 2). In addition, the results also showed that there was a negative and significant correlation between nasal FENO and nasal peak flows (Table 3 and Figure 1a,b). Obviously, exhaled NO concentration is inversely proportional to the airflow rate. FENO measured in healthy subjects with a flow rate of 50 mL/s had a bronchial FENO level of 5–20 ppb, whereas alveolar FENO (CANO) measured at a flow rate of 150–350 mL/s had a concentration of FENO less than 5 ppb [19]. In the present study, nasal FENO was measured with a HypairNO device by the aspirating method with a constant flow over time. However, the application of nasal NO measurement in subjects with AR is relatively complex because some authors have shown that nasal FENO could be changed after allergen exposure. Definitely, Ragab et al. [12] reported that nasal FENO, but not oral FENO, was significantly increased in patients with seasonal AR during the pollen season. However, Palm et al. [13] reported no change in nasal NO concentration in patients with AR. It is noteworthy that

in almost all studies where comorbid sinus disease was excluded, patients with AR had higher nasal NO concentrations compared with healthy subjects. This suggests that there are probably two opposed levels that can determine nasal FENO in patients with RA: firstly, NO gas released from the allergic inflammatory nasal mucosa may be increased although the nasal mucosa are swollen at the same time due to the process of inflammation; secondly, the swollen nasal mucosa might lead to blocked nostrils (ostia) and reduce the flow of NO going out of the nasal cavity, where nasal FENO will be measured. However, the sample size of the present study is not large enough to define the nasal FENO cut-off of a large-scale representative population and for subjects with AR associated with other rhino-sinus comorbidities. This issue is also a main limitation of the present study. Therefore, it is necessary to conduct more studies on nasal FENO in subjects with AR for having reference values in the future.

*Sinusitis* **2021**, *5*, x 7 of 8

The present study's results showed that the nasal FENO cut-off for a positive

diagnosis of AR of 794 ppb was the best diagnostic value (Figure 2, Table 4). The results of ROC curve analysis demonstrated that the cut-off of FENO with the highest Youden index was equivalent to the most significant area under the ROC curve of 794 ppb and had a specificity and sensitivity of 96.7% and 92.6%, respectively (Figure 2 and Table 4).

**Figure 3.** Principle of nasal FENO measurement in subjects with AR [3]. AR: allergic rhinitis; FENO: fractional exhaled nitric oxide; NOS: nitric oxide synthase. **Figure 3.** Principle of nasal FENO measurement in subjects with AR [3]. AR: allergic rhinitis; FENO: fractional exhaled nitric oxide; NOS: nitric oxide synthase.

**5. Conclusions**  Nasal FENO is a potential biomarker in the diagnosis of allergic rhinitis. The measure of nasal FENO is a simple, low-cost, and non-invasive technique. In addition to the use of nasal FENO in the management of patients with allergic rhinitis, nasal FENO might be used for screening patients with sinusitis, nasal polyps, primary cilliar dyskinesia, and Covid-19 infection. Hence, more studies in patients with these conditions are needed in The present study's results showed that the nasal FENO cut-off for a positive diagnosis of AR of 794 ppb was the best diagnostic value (Figure 2, Table 4). The results of ROC curve analysis demonstrated that the cut-off of FENO with the highest Youden index was equivalent to the most significant area under the ROC curve of 794 ppb and had a specificity and sensitivity of 96.7% and 92.6%, respectively (Figure 2 and Table 4). However, the sample size of the present study is not large enough to define the nasal FENO cutoff of a large-scale representative population and for subjects with AR associated with other rhino-sinus comorbidities. This issue is also a main limitation of the present study. Therefore, it is necessary to conduct more studies on nasal FENO in subjects with AR for having reference values in the future.

#### **5. Conclusions**

clinical practice to clarify the role of exhaled NO as a relevant biomarker of non-infectious or viral inflammation. **Author Contributions:** Conceptualization, S.D.-Q., T.N.-T.-D., T.N.-V., V.N.-N., L.H.-T., and T.C.; methodology, S.D.-Q., T.N.-T.-D., K.T.-Q., T.T.-T.-T., T.V.-P.-M., Q.V.-T.-T., K.B.-D., V.N.-N., L.H.- T., and T.C.; software, S.D.-Q., T.N.-T.-D., T.T.-T.-T., and T.N.-V.; validation, S.D.-Q., T.N.-T.-D., Nasal FENO is a potential biomarker in the diagnosis of allergic rhinitis. The measure of nasal FENO is a simple, low-cost, and non-invasive technique. In addition to the use of nasal FENO in the management of patients with allergic rhinitis, nasal FENO might be used for screening patients with sinusitis, nasal polyps, primary cilliar dyskinesia, and Covid-19 infection. Hence, more studies in patients with these conditions are needed in clinical practice to clarify the role of exhaled NO as a relevant biomarker of non-infectious or viral inflammation.

authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

T.T.-T.-T., Q.V.-T.-T.,K.B.-D., V.N.-N., and T.C.; formal analysis, S.D.-Q., T.N.-T.-D., K.T.-Q., T.T.- T.-T., T.N.-V., Q.V.-T.-T., and L.H.-T.; investigation, S.D.-Q., T.N.-T.-D., T.T.-T.-T., and T.N.-V.;

T.T.-T.-T., and T.N.-V.; writing—original draft preparation, S.D.-Q., T.N.-T.-D., Q.V.-T.-T.,K.B.-D., V.N.-N., and L.H.-T.; writing—review and editing, S.D.-Q., T.N.-T.-D., Q.V.-T.-T.,K.B.-D., V.N.-N., L.H.-T., T.C.; Visualization, S.D.-Q., T.N.-T.-D., T.T.-T.-T., T.N.-V., K.B.-D., V.N.-N.; Supervision, S.D.-Q., T.N.-T.-D., T.T.-T.-T., T.N.-V., L.H.-T., and T.C.; project administration, S.D.-Q., T.T.-T.-T., T.N.-V., and T.C.; funding acquisition, S.D.-Q., T.N.-T.-D., K.T.-Q., T.T.-T.-T., and T.N.-V. All

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board of Lam Dong Medical

College (NCKH2018\_TTYS\_04.18).

**Author Contributions:** Conceptualization, S.D.-Q., T.N.-T.-D., T.N.-V., V.N.-N., L.H.-T. and T.C.; methodology, S.D.-Q., T.N.-T.-D., K.T.-Q., T.T.-T.-T., T.V.-P.-M., Q.V.-T.-T., K.B.-D., V.N.-N., L.H.-T. and T.C.; software, S.D.-Q., T.N.-T.-D., T.T.-T.-T. and T.N.-V.; validation, S.D.-Q., T.N.-T.-D., T.T.- T.-T., Q.V.-T.-T., K.B.-D., V.N.-N. and T.C.; formal analysis, S.D.-Q., T.N.-T.-D., K.T.-Q., T.T.-T.-T., T.N.-V., Q.V.-T.-T. and L.H.-T.; investigation, S.D.-Q., T.N.-T.-D., T.T.-T.-T. and T.N.-V.; resources, S.D.-Q., T.N.-T.-D., K.T.-Q., T.T.-T.-T. and T.N.-V.; data curation, S.D.-Q., T.N.-T.-D., T.T.-T.-T. and T.N.-V.; writing—original draft preparation, S.D.-Q., T.N.-T.-D., Q.V.-T.-T., K.B.-D., V.N.-N. and L.H.-T.; writing—review and editing, S.D.-Q., T.N.-T.-D., Q.V.-T.-T., K.B.-D., V.N.-N., L.H.-T., T.C.; Visualization, S.D.-Q., T.N.-T.-D., T.T.-T.-T., T.N.-V., K.B.-D., V.N.-N.; Supervision, S.D.-Q., T.N.-T.-D., T.T.-T.-T., T.N.-V., L.H.-T. and T.C.; project administration, S.D.-Q., T.T.-T.-T., T.N.-V. and T.C.; funding acquisition, S.D.-Q., T.N.-T.-D., K.T.-Q., T.T.-T.-T. and T.N.-V. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board of Lam Dong Medical College (NCKH2018\_TTYS\_04.18).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study. Written informed consent has been obtained from the patient(s) to publish this paper.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Review* **Olfactory Disorders in Post-Acute COVID-19 Syndrome**

**Laura Araújo <sup>1</sup> , Vanessa Arata <sup>1</sup> and Ricardo G. Figueiredo 1,2,3,\***


**Abstract:** Altered smell is one of the most prevalent symptoms in acute COVID-19 infection. Although most patients recover normal neurosensory function in a few weeks, approximately one-tenth of patients report long-term smell dysfunction, including anosmia, hyposmia, parosmia and phantosmia, with a particularly notable impact on quality of life. In this complex scenario, inflammation and cellular damage may play a key role in the pathogenesis of olfactory dysfunctions and may affect olfactory signaling from the peripheral to the central nervous system. Appropriate management of smell disturbances in COVID-19 patients must focus on the underlying mechanisms and the assessment of neurosensorial pathways. This article aims to review the aspects of olfactory impairment, including its pathophysiology, epidemiology, and clinical management in post-acute COVID-19 syndrome (PACS).

**Keywords:** olfactory dysfunction; anosmia; post-acute COVID-19

#### **Citation:** Araújo, L.; Arata, V.; Figueiredo, R.G. Olfactory Disorders in Post-Acute COVID-19 Syndrome. *Sinusitis* **2021**, *5*, 116–122. https:// doi.org/10.3390/sinusitis5020012

Academic Editor: Sy Duong-Quy

Received: 3 September 2021 Accepted: 21 September 2021 Published: 24 September 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

#### **1. Introduction**

Coronavirus 2019 (COVID-19) disease emerged in Wuhan, China, and has subsequently spread worldwide. The pathological agent of COVID-19 is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), an enveloped positive single-stranded RNA virus. COVID-19 is a highly transmissible illness with a broad spectrum of clinical manifestations and variable severity degrees depending on age, comorbidities, genetic factors, and basal metabolic index [1,2]. Individuals may present with a wide range of symptoms, including fatigue, headache, difficulty breathing, diarrhea, nausea, vomiting, loss of taste and smell, runny nose, and muscle and body ache, often indistinguishable from most respiratory viral infections [3].

COVID-19 can induce abnormalities in taste and smell perception, in both the acute and chronic phases of the disease. Smell disturbances are described as: anosmia—a total absence of smell; hyposmia—a diminished sense of smell; parosmia—distorted perception of an existing odor; and phantosmia—perception of smell when no odor source is present. These neurosensory changes have a pronounced impact on quality of life, as most experiences are malodorous, particularly in the qualitative dysfunctions (parosmia and phantosmia) [4].

Angiotensin-converting enzyme-2 receptors (ACE-2) and transmembrane protease serine 2 (TMPRSS2), expressed in the cells of the nasal epithelium, are known pathways for SARS-CoV-2 entry to the respiratory system [5]. Inflammation and cellular damage may play a key role in the pathogenesis of qualitative olfactory dysfunctions. After its internalization, the virus induces an inflammatory response, undergoing maturation and replication inside the cell, as well as involving the recruitment of immune cells [6,7]. SARS-CoV-2 can trigger an unbalanced immune response which overloads the targeted tissues with cytokines and T-cell mediated inflammation [6,7]. The damage caused by the latter may affect olfactory signaling from the peripheral to the central nervous system [8,9].

While most survivors will experience a full recovery, follow-up reveals that a high proportion of individuals still report symptoms after the clearance of the acute infection. The terms 'long COVID' (>4 weeks) as well as 'post-acute COVID' (>3 weeks) and 'chronic COVID' (>12 weeks) have been used to describe these ongoing symptoms [8,10].

#### **2. Epidemiology**

Accumulating evidence indicates that altered smell is one of the most prevalent symptoms in acute COVID-19 infection [11]. In self-report studies, the estimated prevalence of olfactory disorders in acute COVID-19 ranged from 5% to 85%, depending on disease severity, and seems to be higher than in other respiratory viral infections. Although most of the patients recover normal neurosensory function in a few weeks, approximately one-tenth of patients reported long-term smell dysfunction, including anosmia, hyposmia, parosmia and phantosmia, with a particularly notable impact on quality of life [12].

Qualitative olfactory dysfunctions are often undervalued in the clinical management of COVID-19 infection and are generally underestimated in observational self-report studies. Individuals may experience a range of persistent and prolonged olfactory sequelae in PACS (Table 1). Continued loss of smell after several weeks was reported in 1.7–29% of patients with COVID-19 requiring hospitalization [13–16]. Disturbed taste and smell were also prevalent after 6 months in approximately one quarter of home-isolated young adults with a milder course of the disease [17]. In a cohort of 467 patients in the United Kingdom followed up at 4–6 weeks, participants with positive SARS-CoV-2 IgM/IgG antibodies reported significantly higher prevalence of longstanding smell loss compared to participants with a negative antibody test, with rates of full resolution of olfactory impairment of 57.7% and 72.1%, respectively [18]. In addition, female individuals were almost 2.5 times more likely to experience persistent smell loss compared to participants of the male sex, and parosmia was also significantly associated with unresolved smell loss at 4 to 6 weeks follow-up [18].

#### **3. Pathophysiogenesis of Olfactory Dysfunction**

SARS-CoV-2's route of infection basically comprises two pathways: through cell entry factors such as angiotensin-converting enzyme 2 (ACE2), transmembrane protease serine 2 (TMPRSS2), and furin, or through an endosomal route that does not require previous cleavage of the spike protein (S). ACE2 can act as a primary receptor, and, after virus attachment, the spike protein in its surface is cleaved and dissociated by furin, after which the subunit S2 is cleaved by TMPRSS2, changing the structure of the S2 subunit, which ultimately leads to membrane fusion and viral RNA transferring to host cell cytoplasm. An alternative pathway can also be initiated by ACE2 binding and the internalization process involving clathrin and cathepsin L, and, in this case, the virus releases its genetic material directly after endocytosis, as an alternative independent from TMPRSS2 to invade cells [19].

After entering the mouth through salivary particles, the virus can infect cells in filiform and vallate papillae, lingual epithelium and taste buds, all cells that express ACE2, starting its replication, which in turn causes taste impairment [19]. Other potential targets for cell infection due to ACE2 are vascular endothelial cells and adipocytes in parotid and salivary glands. The damage in these cells affects both blood and nutritional supplies and, indirectly, it can change taste perception [19].

Upper airway mucosa has nasal goblet and ciliated cells expressing ACE2 and TM-PRSS2, and these respiratory epithelium cell types may have a role in facilitating SARS-CoV-2 infection by storing viral particles [20].

High levels of ACE2 were found in sustentacular cells of the olfactory system, which are in intimate contact with dendrites of olfactory receptor neurons, and also other olfactory epithelium cells such as ductal cells of Bowman's gland, microvillar cells, globose and horizontal basal cells, and olfactory bulb pericytes [19,21]. It is hypothesized that infection of mesenchymal stromal and vascular cells in the nose and bulb and their subsequent inflammation affects the neuronal conduction, reduces nutritional and water supplies and,

therefore, causes the death of olfactory sensory neurons (OSNs) and damage to olfactory bulb function [20] (Figure 1). Although OSNs are surprisingly not an ACE2 expressing tissue, it has already been described that the spike protein can bind to neural cell receptors, possibly due to cell-to-cell transmission through tunneling nanotubes (TNTs), filamentous cellular projections that form a communication and transportation net between cells [19]. disturbances [22]. To sum up, these are the ways through which SARS-CoV-2 may cause olfactory dysfunction: conductive dysfunction, by mechanically blocking smell from reaching neuroepithelium; sensorineural dysfunction by attacking directly or indirectly olfactory neuroepithelium or OSNs; and central dysfunction, by affecting bulb neurons [22].

High levels of ACE2 were found in sustentacular cells of the olfactory system, which are in intimate contact with dendrites of olfactory receptor neurons, and also other olfactory epithelium cells such as ductal cells of Bowman's gland, microvillar cells, globose and horizontal basal cells, and olfactory bulb pericytes [19,21]. It is hypothesized that infection of mesenchymal stromal and vascular cells in the nose and bulb and their subsequent inflammation affects the neuronal conduction, reduces nutritional and water supplies and, therefore, causes the death of olfactory sensory neurons (OSNs) and damage to olfactory bulb function [20] (Figure 1). Although OSNs are surprisingly not an ACE2 expressing tissue, it has already been described that the spike protein can bind to neural cell receptors, possibly due to cell-to-cell transmission through tunneling nanotubes (TNTs), filamentous cellular projections that form a communication and transportation net be-

Neuropilin-1 represents another host factor that facilitates SARS-CoV-2 entry, and its presence was detected in mitral cells of the olfactory bulb, but not in the OE, and the virus may enter the central nervous system (CNS) through retrograde axonal transport from the nasal cavity in a process mediated by ACE2, TMPRSS2 and nicotinic receptors [22]. SARS-CoV-2 uses olfactory neurons to approach the CNS, and similar mechanisms were described for SARS-CoV-1, MERS- CoV, and HCoV-OCR43. The neuronal damage in axons, the death of neurons and microhemorrhages in the bulb may extend the period of smell

*Sinusitis* **2021**, *5*, x FOR PEER REVIEW 3 of 7

tween cells [19].

**Figure 1.** Pathogenesis of olfactory dysfunction. Infection of mesenchymal stromal and vascular cells in the nose and bulb and their subsequent inflammation affect the neuronal conduction, reduce nutritional and water supplies and, therefore, cause the death of olfactory sensory neurons (OSNs) and damage to olfactory bulb function. SARS-COV-2 indicates severe acute respiratory syndrome coronavirus 2; CNS, central nervous system; OB, olfactory bulb; OSN, olfactory sensory neurons**. Figure 1.** Pathogenesis of olfactory dysfunction. Infection of mesenchymal stromal and vascular cells in the nose and bulb and their subsequent inflammation affect the neuronal conduction, reduce nutritional and water supplies and, therefore, cause the death of olfactory sensory neurons (OSNs) and damage to olfactory bulb function. SARS-COV-2 indicates severe acute respiratory syndrome coronavirus 2; CNS, central nervous system; OB, olfactory bulb; OSN, olfactory sensory neurons.

> **4. Smell Dysfunction in PACS**  Smell dysfunction can occur in the context of various infectious viral diseases [23]. Alteration of smell can be categorized into two distinct types: quantitative and qualitative, and subcategorized in total/complete or partial/incomplete, as well as in unilateral or bilateral [23]. Quantitative loss is seen in anosmia and hyposmia, while qualitative loss is noted in parosmia and phantosmia [24]. Neuropilin-1 represents another host factor that facilitates SARS-CoV-2 entry, and its presence was detected in mitral cells of the olfactory bulb, but not in the OE, and the virus may enter the central nervous system (CNS) through retrograde axonal transport from the nasal cavity in a process mediated by ACE2, TMPRSS2 and nicotinic receptors [22]. SARS-CoV-2 uses olfactory neurons to approach the CNS, and similar mechanisms were described for SARS-CoV-1, MERS- CoV, and HCoV-OCR43. The neuronal damage in axons, the death of neurons and microhemorrhages in the bulb may extend the period of smell disturbances [22].

> To sum up, these are the ways through which SARS-CoV-2 may cause olfactory dysfunction: conductive dysfunction, by mechanically blocking smell from reaching neuroepithelium; sensorineural dysfunction by attacking directly or indirectly olfactory neuroepithelium or OSNs; and central dysfunction, by affecting bulb neurons [22].

#### **4. Smell Dysfunction in PACS**

Smell dysfunction can occur in the context of various infectious viral diseases [23]. Alteration of smell can be categorized into two distinct types: quantitative and qualitative, and subcategorized in total/complete or partial/incomplete, as well as in unilateral or bilateral [23]. Quantitative loss is seen in anosmia and hyposmia, while qualitative loss is noted in parosmia and phantosmia [24].

Anosmia and hyposmia can be assessed by running olfactory tests such as "le nez du vin" or "scratch and sniff" pads containing variable odorant samples [23,25]. Notably, hyposmia and anosmia in infectious diseases are distinguished from nasal inflammation by their lack of seasonal variance, and sometimes permanent length of stay [23].

In contrast to common upper airways infection, rhinorrhea or nasal congestion are less associated with anosmia in COVID -19; however, it can affect the central nervous system, as observed in an 18-FDG PET/CT study, in which a reduction in metabolic activity was reported in the left orbitofrontal cortex, and it can be associated with edema of the olfactory

bulb in MRI [26–28]. Anosmia can lead to suspicion of COVID-19 diagnosis, as it can be the only clinical feature present [29].

Hyposmia was reported in a study in Padua as an isolated or more prominent symptom of SARS-CoV-2 infection, often associated with hypogeusia [25]. Hyposmia and parosmia can be persistent olfactory dysfunctions in PACS [28].

Parosmia and phantosmia are distortions in smell perception. Parosmia is a disorder in which an odor is perceived as a different smell, either pleasant—euosmia—or unpleasant troposmia [25]. Troposmia is often referred to as a burned, foul or rotten smell [27]. In an 18-FDG PET/CT study, the activity in the secondary olfactory cortex was preserved in a patient presenting parosmia post anosmia after COVID-19 infection [27]. In another study, reduced olfactory bulb activity was associated with parosmia [27]. Parosmia and anosmia can be related, and loss of smell can evolve into parosmia in the context of SARS-CoV-2 infection [18].

Parosmia can be related to peripheral and central injuries by SARS-CoV-2, since it can affect OSNs and olfactory centers in the bulb [27]. The growth of new olfactory axons can occur in a non-organized manner and, as a consequence, it prolongs parosmia [18]. Data concerning post-infectious parosmia point to a poorer prognostic value towards the recuperation of smell ability, although olfactory training can help in the recovery of smell [18].

In phantosmia, the smell sensation is generated even in the absence of odors [24]. Smell disturbances can affect taste perception [30]. Taste dysfunctions post COVID-19 infection can also be categorized as qualitative or quantitative. Qualitative taste alterations are known as dysgeusia, whereas quantitative ones are referred to as hypogeusia, in which taste is decreased, and ageusia, in which taste sensation is non-existent [20]. The inflammatory response may cause reparable damage to the taste buds, and as a consequence, a short recovery time can be expected. An unbalanced immune response may also be an agent for a bad prognosis in sensory loss, since the T-cell response is present in sialadenitis and xerostomia [20]. Additionally, distortion in chemosensory perception, such as parosmia or dysgeusia, may increase the probability of long-term smell and/or taste loss and longer COVID-19 symptoms [18].

#### **5. Clinical Management Considerations**

Despite overcoming systemic inflammation and respiratory distress in the acute phase of COVID-19 infection, some patients present prolonged inflammation and tissue damage. Appropriate management of smell disturbances in COVID-19 patients must focus on the underlying mechanisms and the assessment of neurosensorial pathways. Although most COVID-19-related acute olfactory dysfunctions improve spontaneously, treatment for persistent smell symptoms may be reasonable when impairment lingers beyond 2 weeks [31]. However, the efficacy of available treatment in PACS remains unclear.

Olfactory rehabilitation has been described as an effective method for restoring the sense of smell in post-infectious olfactory dysfunction (PIOD). Olfactory training is a simple and safe strategy defined as repeated and conscious sniffing of a set of odorants, for 15–20 s each, at least twice a day [32]. Additionally, the conscious focus on odors, in addition to human olfactory ecology including social and physical environment triggers, may effectively stimulate the neurosensorial system and enhance olfactory performance [33]. A retrospective German study of 153 patients with PIOD showed clinically relevant improvements in overall quantitative and qualitative function upon receiving olfactory training (OT). Additionally, the presence of parosmia was associated with significant improvement of olfactory performance after OT [34].

Oral and nasal corticosteroids may be used to control a potential inflammatory component in PIOD; however, current evidence does not support the routine administration of systemic corticosteroids in this scenario due to safety concerns. Additionally, unless inflammatory features in endoscopic or imaging evaluation are detected, it is improbable that corticosteroids would be helpful. [31]. A recent randomized controlled trial (RCT) failed to prove the superiority of mometasone furoate topical nasal therapy over OT in the treatment of post COVID-19 anosmia. [35]. Notably, intranasal corticosteroid therapy in patients with allergic rhinitis and concomitant COVID-19 infection have recently been reviewed in an ARIA-EAACI position paper. These have been shown to be safe and should be considered, at the recommended dose, on a case-by-case basis [36].


**Table 1.** Prevalence of olfactory dysfunction in post-acute COVID-19 syndrome.

COVID-19 indicates coronavirus disease 2019; Time, time to assessment in days; *n*, sample size.

**Author Contributions:** L.A., V.A., R.G.F. contributed equally to this work. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

