Subjective and psychophysical olfactory and gustatory dysfunction among COVID-19 outpatients; short- and long-term results.

BACKGROUND: Olfactory and gustatory dysfunctions are early symptoms of SARS-CoV-2 infection. Between 20-80% of infected individuals report subjective altered sense of smell and/or taste during infection. Up to 2/3 of previously infected experience persistent olfactory and/or gustatory dysfunction after 6 months. The aim of this study was to examine subjective and psychophysical olfactory and gustatory function in non-hospitalized individuals with acute COVID-19 up to 6 months after infection.
METHODS: Individuals aged 18-80-years with a positive SARS-CoV-2 PCR test no older than 10 days, were eligible. Only individuals able to visit the outpatient examination facilities were included. Gustatory function was tested with the Burgharts Taste Strips and olfactory function was examined with the Brief Smell Identifications test (Danish version). Subjective symptoms were examined through an online questionnaire at inclusion, day 30, 90 and 180 after inclusion.
RESULTS: Fifty-eight SARS-CoV-2 positive and 56 negative controls were included. 58.6% (34/58) of SARS-CoV-2 positive individuals vs. 8.9% (5/56) of negative controls reported subjective olfactory dysfunction at inclusion. For gustatory dysfunction, 46.5% (27/58) of positive individuals reported impairment compared to 8.9% (5/56) of negative controls. In psychophysical tests, 75.9% (46/58) had olfactory dysfunction and 43.1% (25/58) had gustatory dysfunction among the SARS-CoV-2 positive individuals at inclusion. Compared to negative controls, SARS-CoV-2 infected had significantly reduced olfaction and gustation. Previously infected individuals continued to report lower subjective sense of smell 30 days after inclusion, whereafter the difference between the groups diminished. However, after 180 days, 20.7% (12/58) positive individuals still reported reduced sense of smell and taste.
CONCLUSION: Olfactory and gustatory dysfunctions are prevalent symptoms of SARS-CoV-2 infection, but there is inconsistency between subjective reporting and psychophysical test assessment of especially olfaction. Most individuals regain normal function after 30 days, but approximately 20% report persistent olfactory and gustatory dysfunction 6 months after infection.

Background

Shortly after the emergence of the novel coronavirus, SARS-CoV-2, olfactory (OD) and gustatory dysfunction (GD) proved to be early symptoms of Coronavirus Disease 2019 (COVID-19) [1]. The prevalence of OD ranges considerably from 5–80% of infected [2, 3]. However two separate meta-analysis show a similar prevalence at around 50% [4, 5]. Several studies have confirmed the relationship between OD and COVID-19, and sudden onset OD was even suggested as a predictor for COVID-19 in otherwise asymptomatic individuals [6, 7]. While OD has proved a frequent symptom of COVID-19, less focus has been put on GD. There have been speculations that GD was merely a side effect to OD, while other reports show a high prevalence and a distinct pathogenesis [8-10]. The pathogenesis for OD in COVID-19 is unknown, but it differs from other respiratory infections by being independent of nasal congestion [11, 12]. Instead, local inflammation caused primarily by the binding of SARS-CoV-2 virus to the ACE-2 and TMPRSS2 receptors in the sustentacular (non-neural) cells of the olfactory epithelium seems crucial [13, 14]. Furthermore, a more aggressive immune response can cause damage to the neuronal cells, prolonging the period of olfactory dysfunction with a risk of permanent anosmia [13]. Recent radiological finding also suggest that post-COVID-19 OD can possibly lead to olfactory bulb atrophy [13, 15]. The duration of OD differs greatly among COVID-19 infected individuals. Many previously infected regain normal olfactory function after a few weeks [16, 17], and some studies report complete recovery after 6 months [16, 18]. However, several other studies report persistent hyposmia 6 months after primary infection in 20–75% [17, 19–22], and up to a quarter of previously infected still experience olfactory dysfunction one year following infection [23-25].

When assessing olfactory and gustatory function, it is important to recognize that subjective reporting of olfactory and gustatory dysfunction generally corresponds poorly to psychophysical test measurements [26-28].

This study aims to compare the degree of subjective and psychophysical GD and OD in individuals with or without SARS-CoV-2 infection from June 2020 –May 2021 and to monitor subjective recovery up to 6 months after infection.

Methods

This study was a longitudinal prospective case-control study of OD and GD in SARS-CoV-2 positive individuals and negative controls.

Subjects

Participants were recruited from the Capital Region and Region Zealand in Denmark from June 2020 to May 2021. Adults 18-80-years old with a positive SARS-CoV-2 Polymerase Chain Reaction (PCR) test less than 10 days old, were included either via information posters at test facilities in the Capital Region and Region Zealand, or via direct contact through the Danish governmental online communication platform ‘E-boks’. Both ways of inclusion led participants to an online REDCap (Research Electronic Data Capture hosted at the Capital Region, Denmark) questionnaire with further information about the study and a simple yes/no question regarding recent loss of smell and taste. Participants were then contacted by a doctor and invited for psychophysical olfactory and gustatory assessment at either Zealand University Hospital, Koege, Nordsjaellands Hospital, Hilleroed or Rigshospitalet, Copenhagen. Only individuals able to visit the outpatient examination facilities were included. Age-matched negative controls were included through test facilities in both the Capital Region and Region Zealand. Participants who could not fully comprehend the questionnaire and examination was excluded.

The study was conducted in accordance with the Helsinki II Declaration. Written and verbal informed consent was obtained from all participants and the protocol was approved by the Danish Regional Ethics Committee (protocol number: SJ-714 and Letter ID 4336732).

Psychophysical tests

SARS-CoV-2 positive individuals were examined in special facilities designed to accommodate the risk of infection transmission. Negative controls were examined at the otorhinolaryngological outpatient clinics of Zealand University Hospital, Koege, Nordsjaellands Hospital, Hilleroed or Rigshospitalet, Copenhagen. Enrollment was performed maximum 10 days after the initial SARS-CoV-2 PCR test. A trained otorhinolaryngologist assessed the nasal and oral cavity and noted any anatomical variations that could influence the sense of smell or taste. Three further swabs from the nasal cavity, the rhinopharynx and the oropharynx respectively, were performed on both cases and controls and sent for SARS-CoV-2 PCR analysis at the department for Clinical Microbiology at Zealand University Hospital to confirm SARS-CoV-2 positivity or negativity. Information regarding previous infections with SARS-CoV-2 was not available.

We tested gustation using the Burgharts Taste Strips (Burghart Messtechnik GmbH), containing the basic flavors: sweet, sour, bitter and salty. Failure to recognize all 4 flavors was considered as gustatory dysfunction. For olfactory assessment, participants completed the Brief Smell Identification Test™ (Sensonics International Cross-Cultural Smell ID Test, version A (validated in Danish) (BSIT A)). The degree of psychophysical OD (BSIT test) was categorized into either normosmia (BSIT score ≥9/12), hyposmia (≤8/12 - ≥6/12) or anosmia (≤5/12).

Psychophysical testing of taste and smell was done only once per participant at enrolment.

Subjective olfactory and gustatory function

Prior to inclusion, participants answered a simple yes/no question regarding olfactory and gustatory dysfunction in the 7 days preceding their answer. At inclusion participants answered an online REDCap questionnaire including general demographic information, previous or current history of OD or GD, if they could smell sweat, perfume and coffee and if they could taste coffee, salt, sweet, bitter and sour (Y/N). Furthermore, participants were asked about any predisposing illnesses or conditions such as, allergy, chronic rhinosinusitis, congested nose and tobacco use. The questionnaire contained additional questions from a validated smell test questionnaire and the nasal symptoms related questions 1–6 from SNOT-22 [29]. Finally, participants were asked to rank their sense of smell and taste on a numeric rank scale (NRS) with scores from 0–10 (10 being normal). Invitations to the online questionnaire was sent via email at the time of inclusion and at days 30, 90 and 180 following inclusion. For non-responders, up to two-reminder emails were sent.

Statistical methods

Median scores for both psychophysical and subjective measurements of OD and GD were calculated. For test of difference of smell and taste test results in cases and controls Pearson chi squared test and Mann-Whitney test were used for categorical and continuous variables, respectively. P-values <0.05 were considered significant. Results for both OD and GD were stratified according to gender. Sensitivity and specificity for psychophysical OD as a predictor for SARS-CoV-2 infections were calculated along with positive predictive value. We used Stata 13 (StataCorp. 2013. Stata Statistical Software: Release 13. College Station, TX: StataCorp LP.).

Results

In total, 58 SARS-CoV-2 PCR positive individuals and 56 negative controls were included. There was no difference in age and gender distribution amongst the two groups (Table 1). Amongst the SARS-CoV-2 positive, 41.4% (24/58) reported nasal congestion and 34.5% (20/58) reported increased nasal drip while only 16.1% (9/56) of negative controls reported congestion and 14.3% (8/56) reported increased nasal drip. The number of daily tobacco users (1 among cases and 3 among controls) was too low to make stratification. Table 1 also shows other factors that could influence OD/GD.

Table 1

Demographic characteristics and risk factors.

	SARS-CoV-2 positive	SARS-CoV-2 negative
Number	58	56
Male n (%)	30 (51.7)	29 (51.8)
Mean age (Years)	43.3	43.9
Reported symptoms and risk factors of OD/GD
Previous reduced olfaction or gustation n/N (%)	2/58 (3.5)	1/56 (1.8)
Daily tobacco use n/N (%)	1/58 (1.7)	3/56 (5.4)
Increased nasal drip during the last 7 days n/N (%)	20/58 (34.5)	8/56 (14.3)
Nasal congestion during the last 7 days n/N (%)	24/58 (41.4)	9/56 (16.1)
Chronic sinusitis or nasal polyps n/N (%)	2/58 (3.5)	3/56 (5.4)
Hay fever during the last 7 days n/N (%)	3/58 (5.2)	4/56 (7.1)
Daily use of nasal steroid spray n/N (%)	2/58 (3.5)	4/56 (7.1)
Daily use of "over-the-counter" decongestant nasal spray n/N (%)	2/58 (3.5)	4/56 (7.1)
ENT assessment
Septal deviation n/N (%)	3/58 (5.2)	2/56 (3.6)
Inflamed nasal mucosa n/N (%)	4/58 (6.9)	2/56 (3.6)

Demographic information and risk factors for reduced sense of smell and taste for SARS-CoV-2 Polymerase Chain Reaction positive cases and negative controls. OD: Olfactory dysfunction, GD: Gustatory dysfunction, ENT: Ear Nose Throat

Olfactory dysfunction

Psychophysical results

BSIT results showed marked differences in smell between SARS-CoV-2 positives and negatives with 55.2% (32/58) and 1.8% (1/56) being anosmic in the two groups, respectively (Table 2). In total, 75.9% (44/58) of SARS-CoV-2 positives had either anosmia or hyposmia. Median BSIT score was significantly lower for SARS-CoV-2 positive compared to negative and did not differ between sex (Table 2). Table 3 shows the test performance of BSIT as a diagnostic test for SARS-CoV-2.

Table 2

Subjective loss of smell and taste and psychophysical olfactory dysfunction and gustatory dysfunction assessment.

	SARS-CoV-2 positive	SARS-CoV-2 negative	P-value
Total number	58	56
Subjective loss of smell at inclusion n (%)	34 (58.6)	5 (8.9)	<0.001
Subjective loss of taste at inclusion n (%)	27 (46.5)	5 (8.9)	<0.001
Brief Smell Identification Test (BSIT)
Anosmia (BSIT 0–5 correct) n (%)	32 (55.2)	1 (1.8)	<0.001
Hyposmia (BSIT 6–8 correct) n (%)	12 (20.7)	11 (19.6)
Normosmia (BSIT 9–12 correct) n (%)	14 (24.1)	44 (78.6)
Median BSIT score (25–75% IQR)	4.5 (1–8)	10 (9–11)	<0.001
Median BSIT score, females (25–75% IQR)	4.5 (2–9.5)	10 (9–11)	<0.001
Median BSIT score, males (25–75% IQR)	4.5 (1–8)	10 (9–11)	<0.001
Median BSIT score for subjective anosmia/hyposmia (25–75% IQR)	4 (1–8)	8 (7–10)	0.038
Median BSIT score for subjective normosmia (25–75% IQR)	7 (3–10)	10 (9–11)	0.002
Burgharts taste strips
Correctly identify 4/4 taste strips n (%)	33/58 (56.9)	42/56 (75)	<0.001
Median taste test (IQR)	4 (3–4)	4 (3.5–4)	0.03
Median taste test, females (IQR)	4 (3–4)	4 (4–4)	0.003
Mean teste test, males (IQR)	4 (2–4)	4 (3–4)	0.57

Subjective loss of smell and taste answered before inclusion and psychophysical olfactory and gustatory assessment. Median scores with Interquartile Ranges (IQR) for the Brief Smell Identification Test (BSIT) and Burgharts taste strips for SARS-CoV-2 positive and negative controls stratified for gender.

Table 3

Subjective and psychophysical test performance.

	Subjective olfactory dysfunction	Psychophysical olfactory dysfunction
Sensitivity	75.9%	58.6%
Specificity	78.6%	91.1%
Positive Predictive value	78.5%	87%

Sensitivity, specificity and positive predictive values of subjective and psychosocial olfactory dysfunction as diagnostic test for SARS-CoV-2 positivity.

Subjective results

The rate of self-reported hyposmia/anosmia among SARS-CoV-2 positives and negative controls who answered the questionnaire can be seen in Table 4. At the initial questionnaire before inclusion, 58.6% (34/58) of SARS-CoV-2 positive individuals reported subjective OD, compared to 8.9% (5/56) of negative controls (Table 2). Six months after primary infection 20.7% (12/58) reported persistent hyposmia/anosmia (Table 4). The test performance of subjective hyposmia/anosmia as a diagnostic test to detect SARS-CoV-2 can be seen in Table 3.

Table 4

Subjective smell and taste reports and numerical rank scale (NRS) score at 0, 30, 90 and 180 days after follow-up.

	SARS-CoV-2 Positive	SARS-CoV-2 Negative
Answer rate
0 days	51/58 (87.9)	44/56 (78.6)
30 days	39/58 (67.2)	31/56 (55.4)
90 days	19/58 (32.8)	25/56 (44.6)
180 days	36/58 (62.1)	20/56 (35.7)
Number and (%) reporting subjective anosmia or hyposmia
0 days	34/51 (66.7)	5/44 (11.4)
30 days	27/38 (71.1)	2/31 (6.5)
90 days	9/19 (47.4)	1/25 (4.0)
180 days	12/36 (33.3)	1/20 (5.0)
Median self-reported smell function from NRS scale 0–10 (IQR)
0 days	3 (1–8)	10 (9.5–10)
30 days	8.5 (7–10)	10 (10–10)
90 days	10 (7–10)	10 (10–10)
180 days	10 (8–10)	10 (9.5–10)
Median self-reported smell function for people with subjective anosmia or hyposmia from NRS scale 0–10 (IQR)
0 days	2,5 (1–5)	3 (1–8)
30 days	8 (6–9)	7.5 (5–10)
90 days	7 (6–9)	8 (8–8)
180 days	7,5 (6–9.5)	7 (7–7)
Number and (%) reporting subjective gustatory dysfunction
0 days	27/50 (54.0)	5/44 (11.4)
30 days	20/39 (51.3)	2/30 (6.7)
90 days	8/19 (42.1)	1/25 (4.0)
180 days	12/36 (33.3)	1/20 (5.0)
Median self-reported taste from NRS scale 0–10 (IQR)
0 days	7 (3–10)	10 (9–10)
30 days	9 (8–10)	10 (10–10)
90 days	10 (8–10)	10 (10–10)
180 days	10 (9–10)	10 (9.5–10)
Median self-reported taste for people with subjective gustatory dysfunction from NRS scale 0–10 (IQR)
0 days	3 (2–7)	4 (1–8)
30 days	8.5 (6.5–10)	7.5 (5–10)
90 days	8 (8–10)	8 (8–8)
180 days	8.5 (8–10)	7 (7–7)

Questionnaire answer rates and subjectively reported smell and taste impairment at inclusion, day 30, 90 and 180 for SARS-CoV-2 positive and negative controls including median smell and taste scores and Inter Quartile Range (IQR), calculated from subjectively reported smell and taste function ranked in a Numeric Rank Scale score (10 being normal). Median results also stratified for subjective olfactory and gustatory D.

The subjective OD score (NRS from 0–10, 0 being anosmia) are depicted in Fig 1. The median subjective OD score for SARS-CoV-2 positive was lower than median scores for negative controls at inclusion and day 30. At day 90 and 180, median scores for cases and controls showed no difference (Table 4).

Fig 1

Subjective smell scores at inclusion, day 30, 90 and 180.

Subjective smell score at inclusion, 30, 90 and 180 days after inclusion, stratified by SARS-CoV-2 status. Numeric Rank Scale where 0 is anosmia and 10 is normal sense of smell.

Gustatory dysfunction

Objective results

In total, 75% (42/56) of negative controls recognized 4/4 taste strips correctly, while only 56.9% (33/58) of SARS-CoV-2 correctly identified all taste strips (Table 2). Median Burkharts taste strip scores were 4 in both SARS-CoV-2 positives and negative controls.

At the initial questionnaire before inclusion, 46.5% (27/58) of SARS-CoV-2 positive participants reported subjective GD compared to 8.9% (5/56) of negative controls. Median subjective NRS taste scores for SARS-CoV-2 positives and negative controls can be seen in Table 4 and Fig 2. Six months after primary infection 20.7% (12/58) reported persistent GD.

Fig 2

Subjective taste scores at inclusion, day 30, 90 and 180.

Subjective taste score at inclusion, 30, 90 and 180 after inclusion, stratified by SARS-CoV-2 status. Numeric Rank Scale where 0 is anosmia and 10 is normal sense of taste.

Discussion

As previously reported [1, 2], the prevalence of OD and GD among SARS-CoV-2 positive individuals varies considerably. In this study, 58.6% of SARS-CoV-2 positive participants reported subjective OD before inclusion, but a higher fraction (75.9%) had either hyposmia or anosmia in objective BSIT tests, which is similar to results from other studies [2]. Likewise, SARS-CoV-2 positive participants had a significantly lower BSIT median score (4.5) than SARS-CoV-2 negatives. Anosmia was seen amongst 55.2% of SARS-CoV-2 positive participants at inclusion, compared to only 1.8% of negative controls. Compared to other studies [30] the median score for BSIT was slightly lower for our control group.

SARS-CoV-2 positive individuals who reported subjective OD had significantly lower BSIT values. This is not surprising, as objective loss of smell could be expected in persons indicating they had lost their sense of smell. However, also SARS-CoV-2 positive individuals who reported normal olfactory function, had loss of smell when measured objectively, suggesting that some individuals fail to recognize their OD.

As previously stated [1], OD has been suggested as an early indicator of SARS-CoV-2 infection. Our results support previous findings, and indicate that subjectively reported OD and GD are less reliable than objective testing as markers of SARS-CoV-2 infection, as 41.4% of SARS-CoV-2 positive individuals did not have subjective loss of smell. This discrepancy is also known from the general population, where psychophysical tests reveal up to 20% of the background population suffer from olfactory dysfunction, but only around 10% report subjective olfactory dysfunction, suggesting many individuals do not have a clear perception of their sense of smell [31]. The large discrepancy between subjective vs. psychophysical assessments underlines the importance of using psychophysical/objective measurements if OD should be used for early SARS-CoV-2 diagnosis. This is also supported by the fact that psychophysical assessment had higher sensitivity (75.9%) compared to subjective measurement (58.6%). However, the specificity for psychophysical assessment was lower compared to subjective assessment. The Positive Predictive value of BSIT as a diagnostic tool for SARS-CoV-2 positives was slightly higher compared to subjective OD. Thus, an individual reporting subjective sudden onset OD would therefore likely be infected with SARS-CoV-2, but the low sensitivity of subjective reporting would mean a large proportion would be overlooked. However, if OD should be used as a predictive marker for SARS-CoV-2, subjective assessment would likely be the only logistically viable solution due to the large amount of patients. It is, however, crucial to recognize that the absence of OD is a poor marker for SARS-CoV-2 negativity.

During follow-up, OD was reported by 71.1% at day 30, and decreased to 33.3% at day 180 for previous SARS-CoV-2 infected, compared to 6.5% at day 30 and 5.0% at day 180 for negative controls. However, the median subjective NRS score for SARS-CoV-2 positives increased from inclusion to day 30. For negative controls, the subjective NRS score remained stable throughout the follow up period. This shows that even though 20.7% of previously infected continue to report OD, the degree of OD was reduced after 30 days. This is consistent with other results [16–22, 32] where OD is seen to reduce after 30 days in previously infected subjects, while up to a quarter of previously infected report persistent olfactory dysfunction. For the group who reported persistent OD, the median score also increased after 30 days, suggesting some degree of improvement despite persistent subjective OD. This can also be seen in Figs 1 and 2, where the amount of SARS-CoV-2 positive individuals reporting low scores are higher at inclusion and day 30 but normalizes at day 90 and 180. While there is still some uncertainty how the long term recovery differs between different strains of SARS-CoV-2 and how vaccinations influences the long term recovery [33], the high rates of persistent olfactory dysfunction is very concerning. A large proportion of the world’s populations is likely previously infected with SARS-CoV-2, and the total number of individuals at risk of persistent chemosensetive dysfunction is enormous. As these individuals face reduces quality of life, and there is no international consensus of treatment, we face a significant public concern [34].

There were no differences between cases and controls regarding nasal septal deviations or self-reported chronic sinusitis, hay fever, daily use of steroid or local decongestant nasal spray. The rate of daily tobacco users was low in both cases and controls. While tobacco use is known to severely reduce olfaction, the number of users was too low to make stratification. SARS-CoV-2 positive individuals had increased levels of nasal congestion and nasal drip, both of which would increase the level of OD/GD due to obstruction of airflow (Table 1). However, the rates of SARS-CoV-2 positive individuals with OD/GD are much higher than the rates of nasal congestion and nasal drip. This supports that COVID-19 related OD is not solely due to blocked nasal flow.

The gold standard for testing olfactory function in the Western World is the Threshold, Discrimination and Identification (TDI) test and The University of Pennsylvania Smell Identification Test (UPSIT). Compared to these tests, the BSIT has reduced sensitivity but is a cost and time-effective method for screening of large groups of patients [30, 35]. Furthermore, due to the contamination risk when testing SARS-CoV-2 positive individuals, reusing test material is problematical, and we find the single use BSIT is preferable. Likewise, threshold taste strips is preferred [36, 37], but the simple 4 flavor taste strips with equal high concentration is a viable solution when screening larger groups.

In our study, participants with a self-reported history of OD/GD was not excluded. Likewise, smokers and individuals with deviated septum were not excluded. While we recognize this means that we cannot conclude SARS-CoV-2 is the cause of OD/GD for people with prior history or risk factors for OD/GD, we partly account for this by having a comparable control group. Furthermore, all statistical analysis were performed where all individuals with prior OD/GD, septal deviation, smoking history was excluded. This did not significantly change the results or conclusions and it was decided to keep them in the final analysis.

Limitations

During inclusion, answers for BSIT, Burgharts taste strips and objective clinical assessment were noted onto an answer sheet. Unfortunately, the correct answer for the 12th BSIT question was not correctly noted on the answer sheet. This meant a case or control who recognized the correct scent would not receive the correct point. We examined this fault by excluding the 12th answer in the statistical analysis. This did not change any of the conclusions of the study, and therefore the 12th answer was kept in the final results.

It is important to recognize that BSIT and other psychophysical tests are not true objective test of an individual’s olfaction. However, true objective test for olfactory dysfunctions, such as olfactory evoked potential, is not ideal for large group of patients and especially not for SARS-CoV-2 infected individuals. Therefore psychophysical testing is often used as a tool for quantifying sense of smell. This quantification is very useful, and often uncovers olfactory dysfunctions not recognized by the patient. However, when interpreting the results one should recognize that answers depend on the individual performing the test.

Our follow up was limited to subjective answers from questionnaires. As already describes, there is a discrepancy between subjective and psychosocial testing, and the long term recovery results should be interpreted with care. The answer rates of the follow-up questionnaires were generally higher for previously SARS-CoV-2 infected individuals. Individuals who experienced OD or GD might have been more inclined to participate and answer the questionnaire, which could possibly lead to inclusion bias. Likewise, participants with persistent OD of GD could be more inclined to answer follow-up questionnaires. This could overestimate the proportion with OD or GD. Furthermore, we have no information about possible SARS-CoV-2 infection or re-infection in the follow-up period.

Recent anecdotal reports from the latest omicron variant outbreak suggest OD/GD might not be as prevalent as with the original Wuhan virus strain. However, as our inclusion period ran one year from June 2020 to May 2021, at least four different strains were recorded in Denmark (Wuhan, Alpha, Beta, and Gamma). However, mass sequencing was not performed at this time, and therefore we have no information regarding the SARS-CoV-2 strains among the infected participants.

Conclusion

In this case-control study of outpatient SARS-CoV-2 positive individuals without severe COVID-19 disease, 75.9% had objective olfactory and gustatory dysfunction in the acute phase of the infection. Slightly less reported acute subjective olfactory (58.6%) and gustatory (46.5%) dysfunction, but this remained over time with 20% still reporting subjective OD and GD six months after primary infection. Our study contributes significantly to the knowledge of OD and GD dysfunction during active infection and up to 6 months after. We tested both objective and subjective OD and GD in verified SARS-CoV-2 positive individuals compared to age-matched controls. The data brings additional information on how to interpret previous studies based solely on subjective examination. Our results show that SARS-CoV-2 positive participants underestimate their subjective OD and GD compared to objective assessment. This underlines the importance of testing, and not just asking, if OD should be used for early detection of SARS-CoV-2. Among SARS-CoV-2 positive participants subjective OD/GD continued to be higher compared to negative controls at 30, 90 and 180 days follow up.

11 Apr 2022

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Reviewer #1: I have read this well-written manuscript with interest. The article prospectively analyzes a series of 58 COVID-19 patients by analyzing objectively (only at T0) and subjectively (with a follow-up of 6 months) the subjects recruited.

In my opinion a number of issues should be resolved before the article can be considered for publication:

1. In the first part of the introduction the references to the articles on the prevalence of OD and GD in general are dated and refer to articles from the first period of the pandemic (Sadeghat et al, Lechien et al.). Use references with broader case series (Lechien et al. 10.1007 / s00405-020-06548-w), with psychophysical studies (Vaira et al. 10.3390/pathogens10010062) and with revision with meta-analysis (Saniasiaya et al. 10.1002 / lary.29286)

2. Lines 55-56: "The pathogenesis for OD in COVID-19 is unknown, but it differs from other respiratory infections by being independent of nasal congestion" please add a reference.

3. Lines 56-61 Concerning the pathogenesis of olfactory disorders: ACE2 and TMPRSS receptors are mainly concentrated in supporting cells (Brann et al. 10.1126 / sciadv.abc5801; Lechien et al. 10.1007 / s12105-020-01212-5) but not in sensory neurons. The involvement of the bulb is suspected only on the basis of radiological case reports (Tan et al. 10.1002 / lary.30078) but not on the basis of histopathological reports (see the article cited by the authors of Khan et al.). The localized damage at the level of the olfactory epithelium is confirmed by all the histopathological reports currently present in the literature (kirschembaum et al. 10.1016 / S0140-6736 (20) 31525-7; Vaira et al. 10.1017 / S0022215120002455) and this dense with the clinical evidence of rapid recovery in most cases. Bulb atrophy could instead be secondary to a reduction in receptor impulses and not the primary cause of the dysfunction.

4. Both in the introduction and in the discussion the authors must acknowledge that robust studies already exist with 6 (Petrocelli et al. 10.1017/S002221512100116X; Hopkins et al. 10.4193/Rhin20.544, Boscolo-Rizzo et al. 10.1093/chemse/bjab006, Taziki Balajelini et al. 10.1017/S0022215121003935; Prem et al. 10.1007/s00405-021-07153-1, Niklassen et al. 10.1002/lary.29383, Otte et al. 10.1080/00016489.2021.1905178) and 12 month follow-up (Vaira et al. 10.1177/01945998211061511; Boscolo-Rizzo et al. 10.4193/Rhin21.249, Boscolo-Rizzo et al. 10.1007/s00405-021-06839-w). The results obtained by the authors need to be discussed in the light of the results of these other studies.

5. Line 66-67 This is incorrect as the study aims to understand the differences between subjective and objective tests during infection and to prospectively monitor recovery for up to 6 months with subjective methods only.

6. Lines 72-75 Have mechanisms to prevent inclusion bias been put in place? Could it be that subjects with olfactory or gustatory dysfunction were more motivated to participate in the study? If so, this must be recognized in the limitations of the study.

7. Were there any exclusion criteria? Were patients with prior known olfactory and gustatory dysfunctions or conditions predisposing to chemosensitive dysfunctions excluded?

8. How were the controls recruited? Did the controls have to be negative at the time of evaluation or have never contracted the infection before? How was negativity ascertained?

9. lines 113-114: hyposmia is a score between 6 and 8.

10. Lines 122-123 Questionnaire response rates at inclusion were 87.9% (51/58) for SARS-CoV-2 positives and 78.6% (44/56) for negative controls. What does it mean? If it means that the answers were 51 out of 58 why the 7 patients and the 12 controls who did not answer the questionnaire were not excluded from the analysis?

11. lines 145-146 if the previous presence of an olfactory or gustatory disturbance was an exclusion criterion for the enrollment of cases, why was it not also for the controls?

12. Has an analysis been carried out of the correlations of possible clinical and epidemiological risk factors with the persistence of chemosensitive disorders?

13. For the discussion please consider point 4.

14. One of the reasons for the different prevalence detected by the objective methods compared to the subjective ones may be that ODs are present in the general population with a frequency of about 20%. For this reason, subjects who do not know they have an OD report a normal sense of smell while a dysfunction is present on psychophysical tests.

15. The finding of a persistence of ODs in one third of cases at 6 months is a very important and worrying fact that deserves further discussion. Such a high prevalence of residual dysfunction, given the high prevalence of the infection in the population, means that we will have a large number of subjects with disabling long-term morbidity. This is even more serious if we think that:

a. chemosensory disturbances are also frequent in reinfections (Lechien et al. 10.1177 / 0145561320970105; Lechien et al. 10.1111 / joim.13259)

b. chemosensory disorders are also frequent in COVID-19 in vaccinated subjects (Vaira et al. 10.1002 / lary.29964)

c. chemosensory disturbances are also frequent with the omicron variant (10.1002 / alr. 22995)

d. persistent chemosensitive disorders are associated with a significant reduction in the quality of life of patients (Vaira et al. 10.3390 / life12020141, Saniasiaya et al. 10.1017 / S0022215121002279).

e. there are no shared guidelines on therapy.

16. In the limitations it must be added that the follow up was performed only with subjective tests which are not reliable in monitoring the recovery. Subjects who have had a great recovery may report a complete recovery while still being hyposmic (10.4193/Rhin21.249)

Reviewer #2: This study describes subjectively and objectively the effects of SARS-COV-2 infection to olfaction and gustation. This is really an interesting topic, however there are no findings to add new knowledge to what we already know. Also there is a major concern related to the exclusion criteria. According to my opinion patients with chronic sinus diseases or septal deviation that we know that permanently causes olfactory disorders (esp. hyposmia), as well as smokers that we know that smoking affects olfactory function should be excluded. Finally the number of patients (58) is small for safe results.

Reviewer #3: Congratulations to the authors for the study.

Below are some recommendations for the study.

Title

1) “Subjective and objective olfactory…” – The right term in not "objective olfactory test" but psychophysical tests. No objective assessment was performed in all fases of this study, please make this more clear.

Methods

Subjects

2) Line 73 – The sample included individuals aged between 18 and 80 years. However, it is known that the olfactory function, as in other sensory systems is impaired with aging. Therefore, it is very common for individuals after the age of 60 to present alterations in their sense of smell. The sample should have a lower age range, perhaps between 18 and 50 years, due to aging. In addition, these individuals did not undergo a smell assessment before being infected with COVID-19. What if, before the infection, they already had a slight or moderate change in the identification of odors, which they had not noticed? Therefore, it is important to review the age group.

3) Line 86 – I suggest removing “Objective tests” and putting “subjective tests”. See below.

4) Lines 95 and 96 – The authors state that the taste test - Burghart's Taste Stips and the olfactory test - Brief Smell 97 Identification Test are objective tests that were performed by an otolaryngologist. However, these tests are not objective, but subjective/psychophysical, as they depend on the individual's response. The only objective olfactory test is the event-related Olfactory Evoked Potential and the Electroolfactogram, which were not performed in the present sample.

5) Line 95 – How are these tests evaluated for normality and degrees of olfactory and gustatory loss? There is no description for the reader throughout the article.

6) Line 99 – No objective, gustatory or olfactory test was performed in this study. I recommend removing that phrase.

7) Line 107 – The authors state that a questionnaire was made to obtain demographic data and questions 1-6 of the SNOT-22 questionnaire were added. Why didn't you complete the SNOT-22 questionnaire, as it is validated and used internationally? It would be interesting and important to have carried out a complete assessment of the quality of life of each participant during the study period.

8) Line 109 – All participants answered this modified questionnaire four times during the study: at baseline, 30, 60, and 90 days? Is it possible to trust that these questionnaires were actually answered at these times since they were sent by e-mail? Were the psychophysical tests redone 30, 60, and 90 days after the first assessment? Or at least at the end of the study? This is not clear from the text.

Statistical methods

9) Line 112 – There are no objective methods for the assessment of smell and taste in this study.

10) Line 113 – The BSIT test is not objective, it is psychophysical.

Results

11) Lines 122 to 127 – I suggest putting this data in a table and drawing attention in writing only to the most important result.

12) Line 128 – The authors have already put in the title of the table what is being exposed here. I recommend removing and adding the caption.

13) In table 1 – it is necessary to place a legend below the table, for the acronyms PCR, OD, and GD and for other information that is necessary for the understanding of the table.

14) In table 1, in the “Reported symptoms and risk factors of OD/GD” part, add the symbol of (n) for the total number of participants and (%) to identify that the data are being presented in percentage.

15) In table 1, in the “Objective assessment” part, I suggest putting “ENT assessment” and also indicating that the data are presented as a percentage.

16) Line 134 – The acronym IQR is cited for the first time, but there is no full description of - Inter Quartile Range. This description will only occur on line 154.

Olfactory dysfunction

17) Lines 131 to 141 – There is no need to write down all the data that is already presented in the table. Comment only the most important result and tell the reader to analyze the data table.

18) Lines 142 and 143 – Table 2: This information would be better as a table title. I suggest improving the title and adding the legend below the table for the acronyms and other information that is necessary for the understanding of the table.

19) Lines 145 to 152 – the results described are in which table or graph? Please put the reference.

20) Lines 151 and 152: Please, make a table with the results of diagnostic tests for sensitivity, specificity, and positive predictive value.

21) Again, I recommend focusing only on the most important result and not writing out all the results that are already in the tables or graphs.

22) Line 157 (Figure 1) - I recommend improving the quality of the figure.

23) Line 161 - remove 'table 3' and leave only the explanation about the table and add the necessary captions.

Gustatory Dysfunction

24) Lines 167 to 175 – I recommend bringing the results of gustatory dysfunction together with the olfactory one so that the reader has table 2 just below for analysis. Do not describe the results already presented in the table in full. And don't forget to indicate the table.

25) Line 176 (Figure 2) - I recommend improving the quality of the figure.

Discussion

26) Line 181 – Replace “objective tests” with “psychophysical tests”.

27) Lines 181 to 185 – It is interesting to start the discussion by stating the main and most important result of the study. The description presented in this paragraph has already been done in the methodology.

28) Line 194 – “(…) suggesting that some individuals fail to recognize their OD.” This is important to point out, as many individuals cannot have a clear perception of how the sense of smell is.

29) In the discussion also review the description of what are subjective and objective tests, because as already mentioned above, all tests performed by the doctor were psychophysical and not objective. The previous questionnaire applied for the selection of participants is a self-report of olfaction.

30) Do not put so many results, as these are already in the tables. Discuss them only and focus only on the most important ones. Discussion is the crucial part to discuss the study data with what there is already literature.

Limitations

31) The authors report important limitations, mainly the bias in filling out the initial questionnaire.

Conclusion

32) Do not put statistical data in the conclusion. And do not summarize the study in this part.

33) The conclusion should be brief and answer your research question/objective. Do not put information that has already been discussed in the article or that is in the methodology.

34) Objective: “This study aimed to examine subjective and objective olfactory and gustatory function in non hospitalized individuals with acute COVID-19 up to 6 months after infection.” Answer in the conclusion: Was there an improvement in olfactory and gustatory function, after 6 months of the initial diagnosis of COVID?

35) Recommendation: send the article to be reviewed by a native speaker of English.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Submitted filename: Revision doc.pdf

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7 Jul 2022

Reviewer comments Author reply Changes made

1. In the first part of the introduction the references to the articles on the prevalence of OD and GD in general are dated and refer to articles from the first period of the pandemic (Sadeghat et al, Lechien et al.). Use references with broader case series (Lechien et al. 10.1007 / s00405-020-06548-w), with psychophysical studies (Vaira et al. 10.3390/pathogens10010062) and with revision with meta-analysis (Saniasiaya et al. 10.1002 / lary.29286)

Thank you for this excellent comment. We have added references for two separate meta-analysis. Line 50-52: Added: (1). However, the prevalence of OD ranges considerably from 5-80% of infected. (2,3). However, two separate meta-analysis show a similar prevalence at around 50%

2. Lines 55-56: "The pathogenesis for OD in COVID-19 is unknown, but it differs from other respiratory infections by being independent of nasal congestion" please add a reference.

Thank you for pointing out this unreferenced claim. Relevant references have been added. Inserted following references

Lee SH, Yeoh ZX, Sachlin IS, Gazali N, Soelar SA, Foo CY, m.fl. Self-reported symptom study of COVID-19 chemosensory dysfunction in Malaysia. Sci Rep. 8. februar 2022;12(1):2111.

12. Salmon Ceron D, Bartier S, Hautefort C, Nguyen Y, Nevoux J, Hamel AL, m.fl. Self-reported loss of smell without nasal obstruction to identify COVID-19. The multicenter Coranosmia cohort study. J Infect. oktober 2020;81(4):614–20.

3. Lines 56-61 Concerning the pathogenesis of olfactory disorders: ACE2 and TMPRSS receptors are mainly concentrated in supporting cells (Brann et al. 10.1126 / sciadv.abc5801; Lechien et al. 10.1007 / s12105-020-01212-5) but not in sensory neurons. The involvement of the bulb is suspected only on the basis of radiological case reports (Tan et al. 10.1002 / lary.30078) but not on the basis of histopathological reports (see the article cited by the authors of Khan et al.). The localized damage at the level of the olfactory epithelium is confirmed by all the histopathological reports currently present in the literature (kirschembaum et al. 10.1016 / S0140-6736 (20) 31525-7; Vaira et al. 10.1017 / S0022215120002455) and this dense with the clinical evidence of rapid recovery in most cases. Bulb atrophy could instead be secondary to a reduction in receptor impulses and not the primary cause of the dysfunction.

Thank you for this interesting point. Indeed, you are correct that bulb atrophy seems secondary to olfactory dysfunction. We have changed the description of olfactory bulb involvement to avoid confusion. Line 62. “is related to” has been changes to “can possibly lead to” to better describe the involvement of the olfactory bulb.

4. Both in the introduction and in the discussion the authors must acknowledge that robust studies already exist with 6 (Petrocelli et al. 10.1017/S002221512100116X; Hopkins et al. 10.4193/Rhin20.544, Boscolo-Rizzo et al. 10.1093/chemse/bjab006, Taziki Balajelini et al. 10.1017/S0022215121003935; Prem et al. 10.1007/s00405-021-07153-1, Niklassen et al. 10.1002/lary.29383, Otte et al. 10.1080/00016489.2021.1905178) and

12 month follow-up (Vaira et al. 10.1177/01945998211061511; Boscolo-Rizzo et al. 10.4193/Rhin21.249, Boscolo-Rizzo et al. 10.1007/s00405-021-06839-w). The results obtained by the authors need to be discussed in the light of the results of these other studies.

Thank you for these excellent references concerning duration of olfactory dysfunction. We have incorporated them and expanded the background section. Line 64-68: Added information regarding studies examining duration of dysfunction after 6 and 12 month. Furthermore, see questions 13 for changes to the discussion

5. Line 66-67 This is incorrect as the study aims to understand the differences between subjective and objective tests during infection and to prospectively monitor recovery for up to 6 months with subjective methods only.

We appreciate this comment and acknowledge it can be interpreted as we test both subjective and objective for the follow up period. Line 72-74. Deleted “short and long term”

Added: and to monitor subjective recovery up to 6 months after infection.

6. Lines 72-75 Have mechanisms to prevent inclusion bias been put in place? Could it be that subjects with olfactory or gustatory dysfunction were more motivated to participate in the study? If so, this must be recognized in the limitations of the study.

Thank you for this very relevant comment. Indeed, we do have a risk of inclusion bias in this study, and the prevalence of olfactory and gustatory dysfunction would be better examined in a cross-sectional design. We fully acknowledge the risk of bias, and have added a line in the “limitation” section to clarify. Line 282: Added “which could possibly lead to inclusion bias” to clarify

7. Were there any exclusion criteria? Were patients with prior known olfactory and gustatory dysfunctions or conditions predisposing to chemosensitive dysfunctions excluded?

Thank you very much for this excellent comment. Participants were asked about prior olfactory or gustatory issues. However, we chose not to exclude participants with prior issues to better represent the background population, where chemosensitive disorders will also be prevalent. We recognize that this means we cannot conclude that reduced scores in psychophysical tests in SARS-CoV-2 positive individual with prior chemosensitive disorder is caused solely by SARS-CoV-2. However, by having a comparable control group we partly account for this issue and can still conclude that olfactory and gustatory dysfunction are more common amongst SARS-CoV-2 positive individuals. To better explain and highlight this we have added the few exclusion criteria and added the amount of participants who reported previous reduced sense of smell.

Furthermore, we have made a further analysis, where we excluded all smokers, people with deviated septum and participants who answered they had a history of reduced smell or taste. This did not significantly change any results or conclusions. This information has been added in the discussion. Line 89: Added: Participants who could not fully comprehend the questionnaire and examination was excluded.

Line 278: In our study, participants with a self-reported history of OD/GD was not excluded. Likewise, smokers and individuals with deviated septum were not excluded. While we recognize this means that we cannot conclude SARS-CoV-2 is the cause of OD/GD for people with prior history or risk factors for OD/GD, we partly account for this by having a comparable control group. Furthermore, all statistical analysis were performed where all individuals with prior OD/GD, septal deviation, smoking history was excluded. This did not significantly change the results or conclusions and it was decided to keep them in the final analysis.

Table 1: Added column regarding previous olfactory/gustatory dysfunction.

8. How were the controls recruited? Did the controls have to be negative at the time of evaluation or have never contracted the infection before? How was negativity ascertained?

Controls were also included through PCR-test facilities in the two regions. All participants had three further PCR test from different anatomical localizations at inclusion to assert positivity/negativity.

Unfortunately, we did not have any information regarding previous infection with SARS-CoV-2. Line 101: added “on both cases and controls” to clarify.

Line 103: Information regarding previous infections with SARS-CoV-2 was not available.

9. lines 113-114: hyposmia is a score between 6 and 8.

Thank you for the comment. The range has been corrected. Line 108-110: The degree of psychophysical OD (BSIT test) was categorized into either normosmia (BSIT score ≥9/12), hyposmia (≤8 - ≥6) or anosmia (≤5/12).

Per request of reviewer 2, the section has been moved to a different subheading.

10. Lines 122-123 Questionnaire response rates at inclusion were 87.9% (51/58) for SARS-CoV-2 positives and 78.6% (44/56) for negative controls. What does it mean? If it means that the answers were 51 out of 58 why the 7 patients and the 12 controls who did not answer the questionnaire were not excluded from the analysis?

Thank you for making this point. As you propose, the answer rate means that 7 patients and 12 controls did not complete the online questionnaire. However, all participants completed the objective assessment and psychophysical tests. We therefore report results for all patients and follow-up results for the exact number who answered the questionnaire (table 4) No changes made

11. lines 145-146 if the previous presence of an olfactory or gustatory disturbance was an exclusion criterion for the enrollment of cases, why was it not also for the controls?

Thank you for your comment. Please see answer 7 where a detailed explanation is given regarding exclusion. No changes made.

12. Has an analysis been carried out of the correlations of possible clinical and epidemiological risk factors with the persistence of chemosensitive disorders?

Thank you for this excellent comment. It would be very interesting to examine risk factors for persistent olfactory/gustatory dysfunction. However, as the number of participants who completed the follow-up questionnaire was quite low, we concluded that subgroup analysis would have a high risk of bias and not give any valid results.

13. For the discussion please consider point 4.

Again, we thank you for these great references. We have added them in the discussion and expanded the discussion. Line: 252-253: Added references and “while, up to a quarter of previously infected report persistent olfactory dysfunction.”

14. One of the reasons for the different prevalence detected by the objective methods compared to the subjective ones may be that ODs are present in the general population with a frequency of about 20%. For this reason, subjects who do not know they have an OD report a normal sense of smell while a dysfunction is present on psychophysical tests.

Thank you for this excellent point. We completely agree that unrecognized olfactory dysfunction is present in the background population as well. We have added a sentence in the discussion related to this point to clarify. Line. 232-235: Added: “This discrepancy is also known from the general population, where psychophysical tests reveal up to 20% of the background population suffer from OD, but only around 10% report subjective OD”

15. The finding of a persistence of ODs in one third of cases at 6 months is a very important and worrying fact that deserves further discussion. Such a high prevalence of residual dysfunction, given the high prevalence of the infection in the population, means that we will have a large number of subjects with disabling long-term morbidity. This is even more serious if we think that:

a. chemosensory disturbances are also frequent in reinfections (Lechien et al. 10.1177 / 0145561320970105; Lechien et al. 10.1111 / joim.13259)

b. chemosensory disorders are also frequent in COVID-19 in vaccinated subjects (Vaira et al. 10.1002 / lary.29964)

c. chemosensory disturbances are also frequent with the omicron variant (10.1002 / alr. 22995)

d. persistent chemosensitive disorders are associated with a significant reduction in the quality of life of patients (Vaira et al. 10.3390 / life12020141, Saniasiaya et al. 10.1017 / S0022215121002279).

e. there are no shared guidelines on therapy.

Thank you for these interesting points. We completely agree that the number of possible individuals is overwhelming, and recognize we have not fully discussed this. We have added a paragraph in the discussion to highlight this public health concern. Line 257-262: Added: “While there is still some uncertainty how the long term recovery differs between different strains of SARS-CoV-2 and how vaccinations influences the long term recovery (33), the high rates of persistent olfactory dysfunction is very concerning. A large proportion of the world’s populations is likely previously infected with SARS-CoV-2, and the total number of individuals at risk of persistent chemosensetive dysfunction is enormous. These individuals face a reduced quality of life, and this is of significant public concern. Consensus on treatment guidelines are lacking”

16. In the limitations it must be added that the follow up was performed only with subjective tests which are not reliable in monitoring the recovery. Subjects who have had a great recovery may report a complete recovery while still being hyposmic (10.4193/Rhin21.249) We highly appreciate the reviewer has pointed out this weakness in our study. We agree that this design is not optimal to monitor recovery. We have added a paragraph under the limitation section. Line 297-299: Added: “Our follow up was limited to subjective answers from questionnaires. As already described, there is a discrepancy between subjective and psychophysical testing, and the long term recovery results should be interpreted with care.”

Reviewer #2:

This study describes subjectively and objectively the effects of SARS-COV-2 infection to olfaction and gustation. This is really an interesting topic, however there are no findings to add new knowledge to what we already know. Also there is a major concern related to the exclusion criteria. According to my opinion patients with chronic sinus diseases or septal deviation that we know that permanently causes olfactory disorders (esp. hyposmia), as well as smokers that we know that smoking affects olfactory function should be excluded. Finally the number of patients (58) is small for safe results. This case-control study aimed to describe OD in a population-based setting in persons showing up in the public test facilities and who did not need hospital care. We believe this approach adds new information and that our study also adds to some other newly reported findings. We chose that previous illness and relevant co-morbidities were registered for all participants instead of excluding individuals with septal deviation etc. We argue the case-control design allows for this approach but also performed separate analysis where individuals with previous chemosensitive disorders, septal deviation which did not significantly change the results. A paragraph have been added in the discussion. No changes in the manuscript

Line 278: In our study, participants with a self-reported history of OD/GD was not excluded. Likewise, smokers and individuals with deviated septum were not excluded. While we recognize this means that we cannot conclude SARS-CoV-2 is the cause of OD/GD for people with prior history or risk factors for OD/GD, we partly account for this by having a comparable control group. Furthermore, all statistical analysis were performed where all individuals with prior OD/GD, septal deviation, smoking history was excluded. This did not significantly change the results or conclusions and it was decided to keep them in the final analysis.

Reviewer #3:

Congratulations to the authors for the study.

Below are some recommendations for the study.

Title

Thank you for the kind remarks

1) “Subjective and objective olfactory…” – The right term in not "objective olfactory test" but psychophysical tests. No objective assessment was performed in all fases of this study, please make this more clear.

We appreciate this comment very much, as it does indeed suggest we performed true objective testing. We agree that psychophysical test is a more accurate term and have corrected the term throughout the article. Title: Changed “objective” to “psychophysical”

Methods

Subjects

2) Line 73 – The sample included individuals aged between 18 and 80 years. However, it is known that the olfactory function, as in other sensory systems is impaired with aging. Therefore, it is very common for individuals after the age of 60 to present alterations in their sense of smell. The sample should have a lower age range, perhaps between 18 and 50 years, due to aging. In addition, these individuals did not undergo a smell assessment before being infected with COVID-19. What if, before the infection, they already had a slight or moderate change in the identification of odors, which they had not noticed? Therefore, it is important to review the age group.

Thank you for this relevant comment. We have by the case-control design with age-matching of positive and negative persons attempted to overcome the mentioned problems. Changes in manuscript line 88 “Age-matched” is inserted and lines 89-90

3) Line 86 – I suggest removing “Objective tests” and putting “subjective tests”. See below.

See answer 1. Line 70 Changes “objective” to “psychophysical”

4) Lines 95 and 96 – The authors state that the taste test - Burghart's Taste Stips and the olfactory test - Brief Smell 97 Identification Test are objective tests that were performed by an otolaryngologist. However, these tests are not objective, but subjective/psychophysical, as they depend on the individual's response. The only objective olfactory test is the event-related Olfactory Evoked Potential and the Electroolfactogram, which were not performed in the present sample.

See answer 1 Line 94 Changes “objective” to “psychophysical”

5) Line 95 – How are these tests evaluated for normality and degrees of olfactory and gustatory loss? There is no description for the reader throughout the article.

We thank you for pointing this out. Initially, we state the scores under “statistical methods”, but see that it is better suited to follow the description of the different tests. We have moved the description and added a short description regarding Burgharts taste test. Line 106: Failure to recognize all 4 flavors was considered as gustatory dysfunction.

Line 106-108: Moved the following section “The degree of psychophysical OD (BSIT test) was categorized into either normosmia (BSIT score ≥9/12), hyposmia (≤8 - ≥6) or anosmia (≤5/12).”

6) Line 99 – No objective, gustatory or olfactory test was performed in this study. I recommend removing that phrase.

We agree. See answer 1

7) Line 107 – The authors state that a questionnaire was made to obtain demographic data and questions 1-6 of the SNOT-22 questionnaire were added. Why didn't you complete the SNOT-22 questionnaire, as it is validated and used internationally? It would be interesting and important to have carried out a complete assessment of the quality of life of each participant during the study period.

Thank you very much for this point. It would have been very interesting to evaluate quality of life throughout the study period. However, as we also included several other questions, we feared the questionnaires would be too comprehensive and we feared this would deter participants from completing all follow-up questionnaires. As such, we chose only to include the nasal symptoms related questions from SNOT-22 Line 119: Added “The nasal symptoms related”

8) Line 109 – All participants answered this modified questionnaire four times during the study: at baseline, 30, 60, and 90 days? Is it possible to trust that these questionnaires were actually answered at these times since they were sent by e-mail? Were the psychophysical tests redone 30, 60, and 90 days after the first assessment? Or at least at the end of the study? This is not clear from the text. We thank you very much for this comment. The questionnaires itself was hosted online through REDCap, and the email only serves as an invitation with a link. Therefore, we have date and time for all answers. The follow-up was only subjective answers. We have highlighted this in the method section to clarify. Line 73-74: and to monitor subjective recovery up to 6 months after infection.

Statistical methods

9) Line 112 – There are no objective methods for the assessment of smell and taste in this study. We agree. Please see answer 1 No changes in the manuscript

10) Line 113 – The BSIT test is not objective, it is psychophysical. See answer 1 No changes in the manuscript

Results

11) Lines 122 to 127 – I suggest putting this data in a table and drawing attention in writing only to the most important result. We thoroughly appreciate the review of the results section. Overall, we agree that there are many results written in the text and have followed your suggestion to only bring forward the most interesting results. Line 133: Deleted section and replaced with “can be seen in Table 3”

12) Line 128 – The authors have already put in the title of the table what is being exposed here. I recommend removing and adding the caption.

We appreciate the comments on table 1 from the reviewer. We agree that the title is quite long. We have reduced the length and instead put the description in the caption. Table 1: Corrected title. Added “and risk factors for reduced sense of smell and taste for SARS-CoV-2 PCR positive cases and negative controls.” In caption

13) In table 1 – it is necessary to place a legend below the table, for the acronyms PCR, OD, and GD and for other information that is necessary for the understanding of the table.

Thank you for pointing out these acronyms, which are not explained in the table. We have added a description in the caption Table 1. Added: “OD: Olfactory dysfunction, GD: Gustatory dysfunction, ENT: Ear Nose Throat” in caption.

14) In table 1, in the “Reported symptoms and risk factors of OD/GD” part, add the symbol of (n) for the total number of participants and (%) to identify that the data are being presented in percentage.

Thank you very much for point out the faulty table. We have followed your advice to clarify. Table 1: Added n/N (%) throughout

15) In table 1, in the “Objective assessment” part, I suggest putting “ENT assessment” and also indicating that the data are presented as a percentage.

Thank you for this comment. We agree that the term objective can be ambiguous and have replaced it with “ENT”. Table 1: Deleted “Objective”. Added “ENT”

16) Line 134 – The acronym IQR is cited for the first time, but there is no full description of - Inter Quartile Range. This description will only occur on line 154. Thank you for the comment. You are correct that the description is written to late. Due to reduction in the result section, the description is now mentioned at the first use. As suggested, results are now mainly reported in tables and IQR is not mentioned in text. In tables, IQR is written out.

Olfactory dysfunction

17) Lines 131 to 141 – There is no need to write down all the data that is already presented in the table. Comment only the most important result and tell the reader to analyze the data table. We thank the reviewer very much for the comments regarding the result section. We agree the section is extensive and have shortened the whole section. Line 151-156 deleted.

18) Lines 142 and 143 – Table 2: This information would be better as a table title. I suggest improving the title and adding the legend below the table for the acronyms and other information that is necessary for the understanding of the table.

We thank you for pointing out the poor title of the table. We have corrected both title and caption of the table. Table 2: Changes title and caption

19) Lines 145 to 152 – the results described are in which table or graph? Please put the reference.

Thank you for pointing out the missing reference. We have added the required information. Line 1181: Added: Table 4

20) Lines 151 and 152: Please, make a table with the results of diagnostic tests for sensitivity, specificity, and positive predictive value.

We thank you for suggesting making another table to fit these results. We agree that it helps with the understanding. Inserted result into table 3

21) Again, I recommend focusing only on the most important result and not writing out all the results that are already in the tables or graphs.

Again, we thank you for pointing out the extensive result section. As already described, we have shortened the section throughout. Line174-179: Deleted results and referred to table instead.

Line 152-156: deleted results and referred to table 2.

Line 180-181 deleted results and referred to table 2.

22) Line 157 (Figure 1) - I recommend improving the quality of the figure.

Thank you for pointing out the quality of the figure is poor. We suspect the image quality reduced during conversion to the required format. We will contact the journal to resubmit figures with better quality. No changes made.

23) Line 161 - remove 'table 3' and leave only the explanation about the table and add the necessary captions.

Gustatory Dysfunction

Thank you for this comment regarding table 3, now table 4. We have edited the legend of the table. Furthermore, we discovered some inconsistencies in the subheadings and have corrected them. Table 4: Corrected subheading and legend.

24) Lines 167 to 175 – I recommend bringing the results of gustatory dysfunction together with the olfactory one so that the reader has table 2 just below for analysis. Do not describe the results already presented in the table in full. And don't forget to indicate the table.

We thank you for this relevant comment, and agree the table separates the section. However, the journal guidelines suggest inserting the table after the first mention. However, I suspect the table placement will differ in the final print.

We have shortened the result section and instead refer to the relevant tables. Line 204-207: Deleted results. Added “and negative controls can be seen in table 4 and figure 2”

25) Line 176 (Figure 2) - I recommend improving the quality of the figure.

See answer 22. No changes made

Discussion

26) Line 181 – Replace “objective tests” with “psychophysical tests”.

See answer 1 Deleted due to answer 27.

27) Lines 181 to 185 – It is interesting to start the discussion by stating the main and most important result of the study. The description presented in this paragraph has already been done in the methodology.

Thank you for this excellent point. We agree that the discussion is best started off with key results, and the current start has already been mentioned. We have deleted the section. Line 213-217: deleted

28) Line 194 – “(…) suggesting that some individuals fail to recognize their OD.” This is important to point out, as many individuals cannot have a clear perception of how the sense of smell is. We are highly appreciative of this comment. We agree that some may have poor perception of their own sense of smell. We have already added a sentence to accommodate a comment from reviewer 1 and think this elaboration also describes you comment. Line 233-235: Added “This discrepancy is also known from the general population, where psychophysical tests reveal up to 20% of the background population suffer from Olfactory dysfunction, but only around 10% report subjective olfactory dysfunction, suggesting many individuals do not have a clear perception of their sense of smell”

Limitations

29) In the discussion also review the description of what are subjective and objective tests, because as already mentioned above, all tests performed by the doctor were psychophysical and not objective. The previous questionnaire applied for the selection of participants is a self-report of olfaction.

We highly appreciate this comment regarding subjective and objective testing, and agree it is important to discuss the limitation. We have added a section under “limitations”. Line 290-296: added “It is important to recognize that BSIT and other psychophysical tests are not true objective test of an individual’s olfaction. However, true objective test for olfactory dysfunctions, such as olfactory evoked potential, is not ideal for large group of patients and especially not for SARS-CoV-2 infected individuals. Therefore psychophysical testing is often used as a tool for quantifying sense of smell. This quantification is very useful, and often uncovers olfactory dysfunctions not recognized by the patient. However, when interpreting the results one should recognize that answers depend on the individual performing the test.”

30) Do not put so many results, as these are already in the tables. Discuss them only and focus only on the most important ones. Discussion is the crucial part to discuss the study data with what there is already literature.

Thank you again for point out our result and discussion section often repeat results. We completely agree that it is better to refer to tables instead. We have shortened the discussion section accordingly. Line 240: Deleted “78.5% and 87%.”

Line 248-249: Deleted results

31) The authors report important limitations, mainly the bias in filling out the initial questionnaire. Indeed, this study does have limitation, and as you describe, inclusion bias is an issue. Per request of reviewer 1, this point was made clearer in the limitation section. Line 301: added “which could possibly lead to inclusion bias”

During the revision, we found an error in the result section and corrected the number. 200: Error in number. Corrected to 56.9% (33/58)

Submitted filename: Response to Reviewers_150622.docx

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20 Sep 2022

Subjective and psychophysical olfactory and gustatory dysfunction among COVID-19 outpatients; Short- and long-term results

PONE-D-22-08137R1

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22 Sep 2022

PONE-D-22-08137R1

Subjective and psychophysical olfactory and gustatory dysfunction among COVID-19 outpatients; Short- and long-term results.

Dear Dr. Jensen:

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