Keywords
COVID-19 - Olfaction Disorders - Postacute COVID-19 Syndrome - Anosmia - Apolipoproteins
E
Palavras-chave
COVID-19 - Transtornos do Olfato - Síndrome de COVID-19 Pós-aguda - Anosmia - Apolipoproteínas
E
INTRODUCTION
Postviral anosmia is a dysfunction of the olfactory system. It constitutes a common
etiology of olfactory dysfunction (OD) among adults, accounting for around 11 to 40%
of cases, with a greater incidence in females. Typically, this condition manifests
between the fourth and eighth decades of life following an upper respiratory tract
infection.[1]
[2] The anosmia represents a frequent manifestation of the coronavirus disease 2019
(COVID-19), with research indicating a range of incidence rates between 11 and 84%
during the acute phase of the illness.[3]
Anosmia may manifest during the acute phase of the illness or beyond 12 weeks from
the onset, either in isolation or accompanied by other symptoms, such as cognitive
impairment, sleep disturbances, and headache.[3]
[4] For instance, a study with 138 outpatients identified that 7.2% of patients assessed
by olfactory tests[5] persisted with smell alterations after 60 days of illness. Furthermore, a prospective
observational study with 4,182 COVID-19 patients identified anosmia as the third most
prevalent symptom in Long-COVID through patient self-report.[6] Hintschich et al. evaluated 303 patients and showed that smell and taste complaints
persisted objectively after 6 months of infection in 18 and 32% of patients respectively.[7] Likewise, a systematic review by Jafar et al. selected studies that evaluated the
recovery of anosmia in patients after 1, 2, and between 3 and 6 months after COVID-19,
showing the persistence of hyposmia in 37.4, 36.7, and 36.5% of patients[8] respectively.
Apolipoprotein E (APOE) is a protein that results from the transcription of the APOE
gene, which presents three frequent allelic variants (E2, E3, and E4).[9] This protein plays a crucial role in the cholesterol metabolism and interacts with
the angiotensin-converting enzyme (ACE2) receptor, a key factor in the binding process
of the viral spike protein, thereby enabling its cellular entry.[9] Previous publications have suggested a possible role of APOE in conferring protection
against COVID-19 or its more severe clinical manifestations.[10]
[11] To our knowledge, no published studies have reported significant correlations between
APOE polymorphisms, olfactory dysfunction, and COVID-19 severity.
We aimed to analyze the epidemiology of OD and its relationship with APOE polymorphisms
in a cohort of Long-COVID patients.
METHODS
Subjects
We conducted a prospective cohort study involving patients being followed up at a
post-COVID neurological outpatient clinic at the Walter Cantídio University Hospital
in Fortaleza, located in the Northeastern region of Brazil. Patient recruitment occurred
between July and August 2020 and was performed in the context of our research group's
ongoing prospective longitudinal investigation.
Eligibility criteria for participation included a confirmed diagnosis of COVID-19
within the preceding 12 months. The study inclusion criteria required that patients
be aged between 18 and 90 years old, with a positive nasal swab RT-PCR or serological
test for COVID, and post-COVID neurological symptoms that persisted for more than
3 months from the onset and were referred to our outpatient clinic. The exclusion
criteria were absence of neurological symptoms, negative test for COVID-19, pregnancy,
need of oxygen support after acute COVID-19 infection, and patients with an operative
approach in the period between the acute infection and the study evaluation.
In this study, OD was defined as a subjective reduction in olfactory function following
a COVID-19 infection and was confirmed by means of a simple olfactory examination
involving the use of coffee as a test odor in both nostrils. The duration of disfunction
was estimated from the patient's clinical history and the day of evaluation. We defined
persistent OD as persistent complaints for more than 3 months following the resolution
of COVID-19 infection. This crossectional study is an integral part of a larger study
that had previously reported data on the cognitive performance of our sample.[12]
The clinical evaluation was conducted by two neurologists, who worked independently
from each other. The clinical evaluation and identification forms were the same for
all patients. Several patient characteristics were recorded during the evaluation,
including age, sex, years of schooling, initial neurological symptoms, hospitalization,
type of COVID-19 test performed, complementary exams, comorbidities, history of alcohol
abuse, and tobacco use. The Medical Research Council (MRC) dyspnea scale was utilized
to assess dyspnea levels before and after the COVID-19 infection. We also looked for
control patients without COVID-19 infection but, unfortunately, the country was undergoing
a severe health crisis during the pandemic, and patients without the disease were
afraid to participate in the research within a hospital environment.
APOE genotyping analysis
The patient's blood specimens were gathered in EDTA containers per the manufacturer's
directives. Subsequently, genomic DNA was derived from leukocytes in peripheral blood
using the Invitrogen commercial PureLink Genomic DNA Mini Kit (ThermoFisher Inc.,
Waltham, MA, USA). The APOE genotypes were ascertained through real-time polymerase
chain reaction (qPCR), utilizing the TaqMan SNP Genotyping Assay (ThermoFisher Inc.)
allelic discrimination system.[13] For this purpose, we utilized probes under the manufacturer's supplied sequences:
C____904973_10 (rs7412) and C___3084793_20 (rs429358), while considering the data
provided in the catalog number 4351379, and similar techniques described in previous
literature. All evaluations were executed using the QuantStudio(Applied Biosystems,
Foster City, CA, USA) real-time PCR platform.
Statistical analysis
Categorical data were presented as absolute counts and percentages. The associations
between categorical data were assessed using chi-square tests. The normality of continuous
data was first checked using the Kolmogorov-Smirnov test. Normally distributed data
were reported as mean ± standard deviation (SD), while non-normally distributed data
were expressed as median and interquartile range. The one-way analysis of variance
(ANOVA) with the Tukey post-test was used for normal data, and the Kruskal-Wallis
test with the Dunn post-test was utilized for non-normal data. The data were analyzed
with the IBM SPSS Statistics for Macintosh (IBM Corp., Armonk, NY, USA) software,
version 23.0. Statistical significance was set at p < 0.05.
RESULTS
A total of 241 individuals were subjected to screening procedures, out of which 20
were deemed ineligible for inclusion in the study (10 for absence of neurological
symptoms and 10 for testing negative for COVID-19). Thus, the final sample size comprised
221 patients, among whom 186 provided blood specimens for APOE genotyping, and all
subsequent analyses were performed on this subset. The patients were evaluated at
an average of 4.5 months after diagnosis.
[Table 1] describes patients' clinical and sociodemographic characteristics dichotomized according
to OD. Patients were predominantly female (65.4%). The OD group was characterized
by younger age and a lower rate of hospitalization during the acute phase of COVID-19
infection, as indicated by statistical analysis (p = 0.003). No significant differences were observed between the OD and non-OD groups
in terms of depression and cognitive impairment.
Table 1
Comparison of demographics and clinical evaluation between patients with and without
olfactory dysfunction
|
Olfactory dysfunction
|
p-value
|
|
No (n = 133)
|
Yes (n = 53)
|
|
Gender: n (%)
|
Female
|
87 (65.4)
|
36 (67.9)
|
0.744*
|
|
Male
|
46 (34.6)
|
17 (32.1)
|
|
Age: mean(±standard deviation)
|
49(±14.4)
|
41.1(±13.4)
|
< 0.001**
|
|
Years of schooling: n (%)
|
0
|
4 (3.0)
|
0
|
0.031*
|
|
1–4
|
6 (4.5)
|
1 (1.8)
|
|
5–8
|
22 (16.5)
|
3 (5.7)
|
|
9–12
|
38 (28.6)
|
11 (20.8)
|
|
> 12
|
63 (47.4)
|
38 (71.7)
|
|
Cognitive impairment: n (%)
|
Normal
|
39 (29.4)
|
22 (41.5)
|
0.110*
|
|
Cognitive decline
|
94 (70.6)
|
31 (58.5)
|
|
Hospitalization: n (%)
|
No
|
86 (64.7)
|
50 (94.3)
|
0.003*
|
|
Yes
|
47 (35.3)
|
3 (5.7)
|
|
Depression: n (%)
|
No
|
117 (88.0)
|
44 (83.0)
|
0.798*
|
|
Yes
|
16 (12.0)
|
9 (17.0)
|
Notes: *Chi-squared test; **Student t-test.
An independent analysis was conducted for the group of 29 patients without APOE genotyping
to determine if the analyzed group was representative of the cohort. Despite the small
sample size, the correlations of age (p = 0.03) and hospitalization (p = 0.02) remained statistically significant.
[Table 2] describes olfactory dysfunction in comparison to patients' APOE polymorphism. In
both groups, the E3/E3 genotype was found to be the most prevalent. No significant
intergroup differences were observed concerning this particular genotype. However,
the frequency of the E4 allele was found to be lower in the OD group, a finding that
reached statistical significance (p = 0.035).
Table 2
Comparison of APOE polymorphism between patients with and without olfactory dysfunction
|
OD
|
p-value*
|
|
No (n = 133)
|
Yes (n = 53)
|
|
APOE: n (%)
|
E2/E2
|
1 (0.8)
|
0
|
0.100
|
|
E2/E3
|
11 (8.3)
|
4 (7.5)
|
|
E2/E4
|
0
|
1 (1.9)
|
|
E3/E3
|
81 (60.9)
|
41 (77.4)
|
|
E3/E4
|
36 (27.1)
|
7 (13.2)
|
|
E4/E4
|
4 (3.0)
|
0
|
|
Alleles: n (%)
|
|
|
|
|
E2
|
No
|
121 (91.0)
|
48 (90.6)
|
0.930
|
|
Yes
|
12 (9.0)
|
5 (9.4)
|
|
E3
|
No
|
5 (3.8)
|
1 (1.9)
|
0.514
|
|
Yes
|
128 (96.2)
|
52 (98.1)
|
|
E4
|
No
|
93 (69.9)
|
45 (84.9)
|
0.035
|
|
Yes
|
40 (30.1)
|
8 (15.1)
|
Abbreviations: APOE; apolipoprotein E; OD, olfactory dysfunction.
Note: *Chi-squared test.
DISCUSSION
This study evaluated OD in an outpatient population with COVID-19. Our patients had
lower hospitalization incidence, were younger, and had a lower APOE E4 allele frequency
when compared with the non-OD group.
In our sample, patients who experienced persistent OD had a lower incidence of hospitalization,
which is consistent with prior research studies.[14]
[15]
[16] Mendonça et al. evaluated 261 patients prospectively and showed that patients with
mild flu-like syndrome had olfactory dysfunction more frequently (OR = 4.63) than
those with severe COVID-19.[14] In a retrospective study, Talavera et al. evaluated 576 patients in whom OD was
associated with lower mortality (OR = 0.180).[15] Furthermore, a systematic review by von Bartheld et al. included 104 studies and
reported a lower prevalence of OD in hospitalized patients.[16]
Our OD group was younger, which is also in agreement with previous studies.[16]
[17] Giacomelli et al. evaluated 59 patients hospitalized with COVID-19, and those with
olfactory and taste disorders were younger (median 56 vs. 66 years, p = 0.035).[18] In a systematic review, besides age, other factors associated with a higher probability
of OD were Caucasian ethnicity and being female.[16]
Our study found that patients with OD had a lower APOE E4 allele frequency. The study
of APOE polymorphism in post-COVID-19 OD patients is essential, since this disfunction
and APOE genotype are known risk factors for neurodegenerative diseases, notably the
Alzheimer disease (AD).[19]
[20] To date, no other study has evaluated such an association. However, Manzo et al.
suggested investigating APOE E4-positive patients, post-COVID-19 hyposmia, and future
neurodegenerative diseases' subsequent onset.[21] This association deserves to be investigated since Dong et al. found an association
between mild cognitive impairment (MCI), the presence of amyloid and neurodegeneration
biomarkers, and OD, postulating this disfunction as a potential biomarker of prodromal
dementia.[22]
Numerous theories have been proposed to explain the onset of OD following COVID-19,
encompassing factors such as obstruction of odorant transit to the olfactory receptors
due to nasal congestion and damage to the olfactory bulb resulting from cytokine release.[23] Nasal congestion was postulated because of the resemblance of OD presence post-COVID-19
and after other viral infections.[24]
[25] For example, Chapurin et al. demonstrated in a case-control study that there was
no significant difference between objective scores in olfactory tests between the
post-COVID-19 OD groups and other viral infections.[25] However, OD patients post-COVID-19 had no symptoms of nasal congestion and rhinorrhea
when compared with other viral infections.[24] Olfactory bulb injury and atrophy were described in COVID-19 patients, possibly
related to astrogliosis and cytokine release in the acute phase of the disease.[26]
[27]
Regarding a possible explanation for the association found between post-COVID-19 OD
and a lower E4 allele frequency, Zhang et al. showed that, among the APOE alleles,
E4 is the one that least inhibits the entry of SARS-CoV-2, thus conferring a greater
risk for severe forms of COVID-19, which was also shown by Kuo et al.[9]
[10] Thus, the lower frequency of E4 found in our OD group may explain the lower severity
found in these same patients.[7]
[28] Conversely, APOE E4 is a well-known risk factor for AD. There is existing neuroimaging
evidence that has documented structural damage to brain tissue in individuals diagnosed
with COVID-19. The affected regions have been identified as those exhibiting functional
connectivity with the primary olfactory cortex, such as the hippocampus, parahippocampal
cortex, and amygdala.[29] These same sites are also greatly affected in patients with AD. Thus, it would be
reasonable to assume that patients with olfactory dysfunction and APOE E4 polymorphism
could be AD patients in the initial stage of the disease.
Several significant limitations are present in our investigation. The absence of a
control group limits the interpretation of our findings. Our study solely recruited
patients with neurological manifestations and approximately 16% of the recruited patients
lack of APOE genotyping and only 19% of the sample was over 60-years-old, which might
lead to selection bias. We did not conduct a standardized olfactory assessment, which
could have enhanced the reliability of our results, primarily in older patients. We
did not have information about SARS-CoV2 genotype in our patients, as previous study
shows that different variants may have different symptoms epidemiology.[30] Lastly, the absence of neuroimaging investigations prevented us from establishing
a correlation between complaints and radiological findings.
Nevertheless, our sample of Long-COVID-19 outpatients is noteworthy, as it highlights
the persistent nature of OD symptoms following the acute phase of the disease, particularly
in individuals with mild manifestations. Additionally, the inclusion of APOE polymorphism
analysis and its potential correlation with other symptoms strengthens our research's
overall contribution.
Within the existing body of literature on COVID-19, anosmia has been linked with favorable
clinical results, whereas APOE E4 has been associated with unfavorable clinical outcomes.
The association between Long-COVID and cognitive decline is still unclear. A study
conducted by Llana et al. applied cognitive tests to 42 individuals with Long-COVID,
pointing out deficits in procedural memory consolidation and in the immediate recall
of declarative information in patients with anosmia compared with patients without
anosmia and a control group. The average age in the study was 43 years.[31] Another study by Pirker-Kees et al. examined the relationship between anosmia and
cognitive impairment in a small sample of 7 patients with Long-COVID, with an average
age of 79 years. Patients with COVID-19 showed significantly lower ability to identify
odors and lower scores on the Montreal Cognitive Assessment (MoCA) compared with healthy
controls, suggesting that olfactory dysfunction may be a clinical biomarker for cognitive
impairment.[32]
Our research contributes novel insights into these associations by identifying the
APOE E4 allele as a possible protective factor against OD. It is of utmost importance
to conduct longitudinal monitoring of these patients and assess biomarkers of neurodegenerative
disorders in their cerebrospinal fluid or plasma to ascertain the persistence of the
impairment in olfactory functioning after a certain timeframe and determine whether
there is any correlation with the onset of neurodegenerative diseases.
Bibliographical Record
Danilo Nunes Oliveira, José Wagner Leonel Tavares-Júnior, Werbety Lucas Queiroz Feitosa,
Letícia Chaves Vieira Cunha, Carmem Meyve Pereira Gomes, Caroline Aquino Moreira-Nunes,
Jean Breno Silveira da Silva, Artur Victor Menezes Sousa, Safira de Brito Gaspar,
Emmanuelle Silva Tavares Sobreira, Laís Lacerda Brasil de Oliveira, Raquel Carvalho
Montenegro, Maria Elisabete Amaral de Moraes, Manoel Alves Sobreira-Neto, Pedro Braga-Neto.
Long-COVID olfactory dysfunction: allele E4 of apolipoprotein E as a possible protective
factor. Arq Neuropsiquiatr 2024; 82: s00441788272.
DOI: 10.1055/s-0044-1788272
