Neurobiological Underpinnings of Neuropsychological Consequences in Long COVID: Exploration of Two Translational Pathways
Conditions: Long COVID
Sponsor: Universiteit Antwerpen

COV-N-PSY | COVID-19 NEURO-PSYCHIATRIC CONSEQUENCES
Neurobiological underpinnings of long COVID neuropsychiatric consequences:
exploration of two translational pathways
Livia De Picker1,3, Katrien Skorobogatov1,3, Filip Van Den Eede2,3, Kurt Beeckmans1,
Greta Moorkens2, Bernard Sabbe3, Manuel Morrens1,3
Protocol version 4.0 - Date:  FEB2022
1 University Psychiatric Hospital Duffel
2 University Hospital Antwerp
3 Collaborative Antwerp Psychiatric Research Institute, University of Antwerp
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Table of Contents
List of abbreviations
1. Introduction
1.1. Neuropsychiatric consequences following COVID-19
1.2.  Neurocognitive deficits following COVID-19
1.3. Immune disturbances as translational pathways
1.4. Systemic immunological disturbance
1.5. HERV-W-ENV reactivation
1.6. Hypothalamo-pituitary-adrenal (HPA) axis function
1.7. Insomnia
2. Objectives
3. Methodology
3.1. Recruitment and study population
3.2. Study design
3.3. Study measurements
3.3.1. Psychiatric interview
3.3.2. Neuropsychological assessment
3.3.3. Blood sampling
3.3.4. Cortisol measurement (HPA-axis)
3.3.5. Electronic clinical measures
3.4. Endpoint measurement
3.5 Statistical analysis
3.6. Management of the database
3.7. Ethical aspects
References
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Summary
The coronavirus disease 2019 (COVID-19), caused by the novel severe acute
respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused more than 120 million
cases and 2.6 million deaths to date, since 2020. As the pandemic spread, there has
been a growing recognition of neuro-psychiatric (NP) impact of COVID infections.
Such complications have been described during the acute phase of the infection
with emerging data showing that, particularly in severe cases of COVID-19 infection,
the acute viral infection is associated with delirium, confusion, and psychosis,
possibly related to the cytokine storm (Rogers et al. 2020). Importantly, a much
larger proportion of even mild COVID-19 cases experience neuropsychiatric
complications at follow-up. Six months after clinical recovery, an estimated 34% of
COVID-19 survivors are diagnosed with new-onset neuropsychiatric disorders, in
particular anxiety disorders (17%), mood disorders (14%), substance use disorders
(7%) and insomnia (5%) (Taquet et al., 2021). It is clear that long COVID NP
symptoms threaten the quality of life and the societal functioning of a significant
proportion of the active population (De Lorenzo et al., 2020; Ma et al., 2020; Mazza
et al., 2020; Varatharaj et al., 2020, Gennaro et al, 2021). It is estimated that this will
severely impact the health of patients infected with COVID-19, long after the
pandemic will have ended thanks to vaccination efforts. Despite the major
socio-economic consequences of the long-term impact of the pandemic, a
standardized way to screen, diagnose, predict and specifically treat patients who
develop or will develop these NP symptoms is currently not available. Despite
post-viral or post-infectious neuropsychiatric symptomatology has been recognized
and studied prior to the COVID-19 pandemic, crucial knowledge about the
neurobiological mechanisms underlying these symptoms is still lacking.
Large population cohort studies have replicated the finding of systemic immune
disturbances in cohorts of patients with depression. Among post-COVID NP
patients, baseline systemic inflammation (systemic immune inflammatory index: SII=
thrombocyte count x neutrophile count / lymphocyte count) predicted severity of
depressive psychopathology at the three-months follow-up (Mazza et al, 2021).
Also, long COVID can be regarded as a condition of chronic stress, which could
theoretically be associated with disturbances of the hypothalamo-pituitary-adrenal
(HPA) axis, although no studies on HPA axis (dys)function in long COVID have been
reported yet.
The aims of this study are to describe the standardized evaluation of the
psychopathological and neurocognitive function of long COVID patients and their
evolution over time; to explore the cross-sectional and longitudinal association
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between long COVID symptoms and immunological measures; and to examine the
neuro-endocrine (HPA-axis) characteristics of long COVID syndrome.
Three participant groups will be included: 1) long COVID patients with
neuropsychological complaints (P), 2) COVID-survivors without persistent complaints
(Ca) and 3) healthy volunteers (Cb).
This is a longitudinal study, starting from November 1st 2021. Enrollment will
continue for an estimated period of 24 months, depending on the time course of
the epidemic curve. Last-patient-in is expected no later than November 1st, 2023.
The study will be organized into three work packages (WP1, WP2 and WP3). WP1
includes psychiatric assessments using questionnaires and blood sampling and is
the standard WP for every participant. WP2 includes cortisol measurement using
saliva sampling for patients and controls from Cb. who do not fulfill specific
exclusion criteria (pregnancy, breastfeeding, hormonal therapy with exception of
contraceptives, cortisol treatment). Finally, WP3 includes an extensive
neurocognitive assessment by a specialized neuropsychologist for both patients and
controls from Ca who do not fulfill specific exclusion criteria (sedative medication,
pre-existing neurological disease, severe substance abuse, and IQ < 90).
Patients and controls from Ca will be referred mainly by the University Hospital of
Antwerp (UZA) and by long COVID patient support groups and enrolled at first visit
to UPCD, where follow-up will also take place at 3, 6 and 12 months. WP1 will be
performed at each timepoint, while WP2 and WP3 will only be carried out at
baseline and 6 months.
Total sample size is estimated on 130 participants.
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List of Abbreviations
-
ACTH: adrenocorticotropic hormone
-
BACS: brief assessment of cognition in schizophrenia
-
BDI: Beck depression index
-
Ca: Control group a: COVID-survivors without NP complaints
-
CAR: cortisol awakening response
-
Cb: Control group b: healthy controls
-
CFS: chronic fatigue syndrome
-
CNS: central nervous system
-
COVID-19: coronavirus disease of 2019
-
CRF: corticotropin-releasing factor
-
CRP: C-reactive protein
-
DNA: deoxyribonucleic acid
-
DRUID: driving under the influence of drugs, alcohol and medicines
-
EDTA: ethylenediaminetetraacetic acid
-
FU: follow-up
-
HDL: high-density lipoprotein
-
HERV-W-ENV: human endogenous retrovirus-W envelope
-
HPA: hypothalamic-pituitary axis
-
IFN-g: interferon gamma
-
IL-1b: interleukin 1-b
-
IL-6: interleukin 6
-
NESDA: Netherlands Study of Depression and Anxiety
-
NP: neuropsychiatric
-
PET: positron emission tomography
-
P: patient group
-
PTSD: post-traumatic stress disorder
-
RT-PCR: reverse transcription polymerase chain reaction
-
SARS-CoV-2: severe acute respiratory syndrome coronavirus 2
-
SII: systemic immune-inflammatory index
-
TG: triglycerides
-
TNF-a: tumor necrosis factor alpha
-
TSPO: translocator protein
-
UPCD: Universitair Psychiatrisch Centrum Duffel (Universitary Psychiatry Hospital of
Duffel)
-
UZA: Universitair Ziekenhuis Antwerpen (Universitary Hospital of Antwerp)
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1.
Introduction
1.1.
Neuropsychiatric consequences following COVID-19
The coronavirus disease 2019 (COVID-19), caused by the novel severe acute
respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused more than 183 million
cases and 3,96 million deaths to date, since 2020 (1). As the pandemic spread, there
has been a growing recognition of neuro-psychiatric (NP) impact of COVID
infections. Such complications have been described during the acute phase of the
infection with emerging data showing that, particularly in severe cases of COVID-19
infection, the acute viral infection is associated with delirium, confusion, and
psychosis, possibly related to the cytokine storm (2). Importantly, a much larger
proportion of even mild COVID-19 cases experience neuropsychiatric complications
at follow-up. Six months after clinical recovery, an estimated 34% of COVID-19
survivors are diagnosed with new-onset neuropsychiatric disorders, in particular
anxiety disorders (17%), mood disorders (14%), substance use disorders (7%) and
insomnia (5%) (3). The NICE guidelines have defined acute COVID-19 as signs and
symptoms
of
COVID-19
from
the
contamination
until
4
weeks,
ongoing
symptomatic COVID-19 from 4 to 12 weeks and post-COVID-19 syndrome for signs
and symptoms which persist for more than 12 weeks (4). These long-term post
COVID NP symptoms (also referred to as “long COVID”, defined as signs and
symptoms of COVID-19 after 4 weeks), which are often accompanied by deficits in
several cognitive domains (including attention) (5), are unrelated to respiratory
insufficiency suggesting independent brain dysfunction. Studies conducted in Italy,
Germany, UK and the US found that post COVID NP symptoms seem to affect
between 20 to 70% of patients infected with COVID-19 and to last months after
respiratory symptoms resolved, suggesting a separate and enduring mechanism
(3,6–11). Most strikingly, these NP complications also occur in younger adults with a
mild acute COVID-19 presentation (9,10,12).
It is clear that long COVID NP symptoms threaten the quality of life and the societal
functioning of a significant proportion of the active population (6,8,13,14). It is
estimated that this will severely impact the health of patients infected with
COVID-19, long after the pandemic will have ended thanks to vaccination efforts.
This may even become a persisting concern if SARS-CoV-2 infection becomes
endemic with a pattern analogous to that of flu with Influenza, in particular as the
risk of a new neuropsychiatric diagnosis following COVID-19 is 44% greater
compared with influenza(3). Despite the major socio-economic consequences of the
long-term impact of the pandemic, a standardized way to screen, diagnose, predict
and specifically treat patients who develop or will develop these NP symptoms is
currently
not
available.
Despite
post-viral or post-infectious neuropsychiatric
symptomatology
has
been
recognized
and studied prior to the COVID-19
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pandemic, crucial knowledge about the neurobiological mechanisms underlying
these symptoms is still lacking.
1.2.
Neurocognitive deficits following COVID-19
Memory and attention problems, reduced information processing and executive
dysfunction are among the most frequent complaints of long COVID patients.
Specific complaints include concentration difficulties, mental exertion, word finding
difficulties, problems with both explicit and procedural memory retrieval (i.e.
difficulty completing tasks which used to be “automatic”) and problems with
planning
and
organization of daily activities, combined with the subjective
experience of “brain fog” (7,15–17).
Scientific evidence is indicating these cognitive deficits are alarmingly widespread
among COVID-survivors. In a longitudinal cohort study of 130 Italian patients aged
18-70 years who were assessed with the Brief Assessment of Cognition in
Schizophrenia (BACS) (18) at one and three months after acute COVID-19
pneumonia, only 25 patients (19%) showed equivalent scores within the normal
range in all cognitive domains (i.e., verbal memory, verbal fluency, working memory,
selective attention, processing speed, psychomotor coordination and planning and
problem solving), whereas 21 (16%) were poor performers in at least one function,
22 (17%) in two, 18 (14%) in three, 14 (11%) in four, 7 (5%) in five, and 2 (1.5%)
demonstrated deficits in all domains (7).
In the study carried out by Miskowiak et al. (17) cognitive functioning was assessed
in a group of 29 Danish COVID-patients approximately three to four months after
hospitalisation by means of the Screen for Cognitive Impairment in Psychiatry (SCIP)
and the Trail Making Test.
Fifty-nine % of the patients showed cognitive deficits
(38% was globally impaired and 21% was selective impaired), with verbal memory
and executive functions being most affected. Ortelli et al. (16) investigated cognitive
functions by means of the MOCA, the FAB and three computerized attentive tasks
(the vigilance task, the Stroop interference task and the Navon task) in a group of 12
Italian COVID-survivors approximately 9 to 13 weeks from onset of infection. In
comparison with a healthy control group, the patient group showed a significant
reduced global cognitive functioning (MOCA and FAB) and significant more
problems with vigilance and executive attention (computerized attentive tasks).
In the study of Mazza et al. (2121), the presence of psychopathology (mainly
depressive symptomatology) was shown to significantly influence neurocognition in
post-COVID patients, most notably affecting verbal fluency, information processing,
and executive functions at one and/or three months assessments. Patients’ sex,
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previous psychiatric diagnosis, and duration of hospitalization for COVID-19 did not
significantly affect the neurocognitive function (7).
Importantly, preliminary findings of the same research group indicate that the
observed cognitive deficits worsen between three- and six-month follow-up (Figure
1, unpublished data). Furthermore, the neurocognitive profile of these patients
demonstrates some similarities with patterns observed in depressed patients using
the same assessment method. Yet whereas the depressed symptoms of post-COVID
NP
patients
seem
to
respond
well to regular treatment with serotonergic
antidepressants, the cognitive symptoms do not.
Figure 1: Cognitive profile of post-COVID patients compared to Healthy controls (HC) and depressed patients
(MDD) (Mazza et al. 2021, in prep) (7)
1.3.
Immune disturbances as translational pathways
1.3.1.
Systemic immunological disturbance
Large population cohort studies have replicated the finding of systemic immune
disturbances (such as but not limited to IL-6 and C-reactive protein (CRP)) in cohorts
of patients with depression (19). Longitudinal studies demonstrated the predictive
value of these immunomarkers towards clinical outcome and the development of
comorbidities such as chronic fatigue, anorexia, insomnia, somatic disease, …
(20–23). Anti-inflammatory agents have been confirmed as valid add-on treatment
options to improve depressive symptoms (24). In longitudinal analyses, higher
baseline IL-6 levels predicted a subsequent chronic course of depression in women
but not in men (25). Similarly, higher baseline IL-6 levels in childhood were
associated with depressive and psychotic symptoms at age 18 (26).
Finally, the clinical observation that some patients with long COVID complaints
experience
an
improvement
of
their
NC
symptoms
following
SARS-CoV-2
reinfection or vaccination supports the hypothesis of immune-related pathways
involved in the pathophysiology of post-COVID NP complications.
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Figure 2: Effect of the decrease of systemic inflammation from hospital admission to 3 months follow-up on the
pattern of change of depressive symptoms (measured with BDI-13) during follow-up. (7)
1.3.2.
HERV-W-ENV reactivation
It is now clear that COVID-19 NP symptoms can persist beyond 6 months
post-infection. Within a window of 90 days, 20% of COVID-survivors are diagnosed
with a new psychiatric disorder, which increases to 34% after 6 months (3,11). This
suggests that a subsequent autonomous response to the initial infection needs to
occur. In this context, a very plausible biological candidate has recently been
identified. Indeed, very recent results showed that COVID-19 activates the human
endogenous retrovirus-W-envelope protein (HERV-W ENV), which is known to
induce persistent innate immune responses and cause neurotoxicity and CNS
dysfunction (27). HERV-W elements are atypical segments in the human genome
originating from retroviruses, which are able to infiltrate the cell nucleus. Although
most HERVs are not transcripted, they encode an envelope protein causing
inflammation and neurotoxicity (27). HERV-W-ENV was found to be highly expressed
in the lymphocytes of acute COVID-19 patients which was correlated with
inflammatory markers (Balestrieri et al, 2021).
Importantly, HERV-W-ENV has previously been implicated in some psychiatric
disorders, such as schizophrenia and bipolar disorder (27,28), as well as in
neurological conditions such as multiple sclerosis. Importantly, in major mood and
psychotic disorders, an inflamed subpopulation represented patients in which the
pathology involved HERV-W ENV protein and was associated with increased serum
pro-inflammatory cytokines (28).
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Another recent publication provided a scientific breakthrough in this domain by
identifying the pathogenic pathways induced by HERV-W ENV through altered
glutamate functioning and to abnormal behavioural patterns (Johansson et al.
2020). Interestingly, this study evidenced the pivotal role of microglial activation in
neurotoxicity and, therefore, highlighted a central common point with the consistent
findings of microglial activation as measured with TSPO PET imaging in depression
(29).
The reactivation of HERV-W-ENV by SARS-CoV-2 in a proportion of COVID-19
survivors may therefore represent the underlying cause of the persistent systemic
inflammation and accompanying neuropsychiatric symptomatology.
1.3.3.
Hypothalamo-pituitary-adrenal (HPA) axis function
Long COVID can be regarded as a condition of chronic stress, which could
theoretically be associated with disturbances of the hypothalamo-pituitary-adrenal
(HPA) axis, although no studies on HPA axis (dys)function in long COVID have been
reported yet.
The HPA axis is activated by both physical and psychological stressors and ends
with the production of cortisol, which participates in the organism’s response to
stress, in order to mobilize energy from storages throughout the body and to
regulate the psychophysiological, immunological and behavioral responses to
stress. The HPA axis activity is regulated by cortisol through negative feedback on
the hypothalamus and the pituitary, decreasing the secretion of CRF and ACTH
respectively (30–33).
Under normal circumstances, cortisol levels vary across the day, with a peak rise
following awakening in the morning (the cortisol awakening response or CAR) and a
gradual decline throughout the day. In patients with chronic fatigue syndrome (CFS),
an attenuated CAR increase and changes in diurnal cortisol slope (DCS) have been
found to be important neuro-endocrine correlates.
Taking the chronic stress and the overlapping symptoms of chronic fatigue and
exhaustion into account, one could expect a hypofunction of the HPA axis in long
COVID, in analogy with the findings in CFS. Furthermore, there may also be an
association with cognitive function and with immunological disturbances (34). Also,
the neuro-endocrine correlates might be predictive for the recovery and the
response to treatment in long COVID patients. Importantly, HPA axis hyperactivity is
well-known to be associated with depression (35).
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1.4.
Insomnia
Genome Wide Association Studies have demonstrated a strong correlation of the
genetic risk of insomnia with depression and anxiety (36,37). Furthermore, insomnia
has been identified as a primary risk factor for the onset, persistence and relapse of
anxiety, depression and PTSD. In a Network Outcome Analysis of 768 individuals
without history of depression, difficulty initiating sleep was one of only five direct
predictors of first-onset major depressive disorder after six years follow-up (38).
Depending on the study methods, in long COVID patients the prevalence of
clinically relevant insomnia was estimated between 5% (diagnosis of insomnia
disorder in electronic medical records <6m following infection) and 24% (self-rated
insomnia symptoms above clinical cut-off at 3-month follow-up). These symptoms
could represent an important risk factor in the subsequent development of anxiety
and depressive disorders, and a key mechanism underlying cognitive deficits.
2.
Research Objectives
1) Describe
the
standardized
evaluation
of
the
psychopathological
and
neurocognitive function of long COVID NP patients and their evolution over
time;
2) Explore the cross-sectional and longitudinal association between long COVID
NP symptoms and immunological measures;
3) Define the prevalence and clinical characteristics of insomnia in long COVID
NP patients, as well as its correlation to the clinical psychiatric and
neurocognitive symptom domains;
4) Compare the immunological profile of long COVID NP patients to those of
existing cohorts of comparable primary psychiatric conditions and healthy
controls;
5) To examine the neuro-endocrine (HPA-axis) characteristics of long COVID
syndrome
3.
Methodology
3.1.
Recruitment and study population
Inclusion criteria - patients:
All patients 18-70 years referred to the University Psychiatric Hospital (Campus UZA
and Campus Duffel) for psychological and/or cognitive complaints at least >4 weeks
following a confirmed diagnosis of COVID-19 infection with a positive PCR test or
an antibodies test will be invited to participate in the study. The sample size of the
patients is estimated on 50 participants. The patients have to be examined by their
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treating physician before enrollment to make sure other medical causes for their
complaints are excluded. A positive score on at least two domains (psychological /
cognitive) is necessary during the screening phase (see 3.3.1).
Inclusion criteria – controls:
Two control groups, one of 30 healthy controls (Cb), another of 50 COVID-19
survivors without persistent complaints (Ca), matched on age, sex and education
with the patient group will be included. The participants for Ca will be recruited in
UZA and by advertising. The healthy controls (Cb) will be recruited by mailing within
the hospital and by advertising. A positive score on maximum one domain
(psychological / cognitive) is allowed in the absence of a psychiatric diagnosis
(confirmed by the MINI) and when the complaint causes significant distress (which is
investigated during the screening phase).
General exclusion criteria:
Participant is unable to read and understand the consent form and patient-reported
outcomes, complete study-related procedures, or communicate with the study staff
and informed consent cannot realistically be obtained in retrospect or with the help
of a competent family member or legal representative.
Exclusion criteria for each subgroup:
-
HPA subgroup
▪
Participant is pregnant or breastfeeding
▪
Participant
receives
hormonal
replacement
therapy
(contraception is allowed).
▪
Participant is treated with cortisol <4 weeks ago
-
Neurocognitive assessment:
▪
IQ < 90 (screened with Raven Standard Progressive Matrices
(Short Form) (RSPM-SF) (39)
▪
Participant takes sedative medication
-
Benzodiazepines: Larger than the equivalent of diazepam
10mg per day. Last administration <8 hours prior to the
neurocognitive tests.
-
New sedative antipsychotics/antidepressants (<4 weeks)
-
Other medication from DRUID class III (<4 weeks), last
administration <8 hours prior to the neurocognitive tests,
or  causing significant sedation.
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▪
Severe substance abuse (alcohol + drugs)
▪
Pre-existing neurological diseases
Figure 3: Flowchart of the COVID-19 participant inclusion and the work packages (WP). Participants with a
confirmed positive PCR test will be included at UZA or UPCD and are classified in the patient group when
suffering from neuropsychiatric symptoms for more than 4 weeks. All participants undergo psychiatric
assessment and blood sampling on every time point of the study (WP1). Additionally, if patients do not fulfill the
exclusion criteria for HPA-axis assessment (pregnancy, breastfeeding, treatment with corticosteroids, hormonal
replacement therapy with exception of contraceptives), they are included in the cortisol subgroup (WP2). Finally,
all participants, except those treated with class III drugs according to the DRUID classification, those suffering
from pre-existing neurological disease or substance abuse and those with an IQ below 90, undergo
neurocognitive assessment (WP3).
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Figure 4: Flowchart of the healthy control inclusion and the work packages (WP). Healthy volunteers can be
included in Cb if they were not infected with Sars-CoV-2 in the past and if they do not complain from psychic or
cognitive symptoms. If they meet the exclusion criteria for the cortisol testing, they are excluded from the study.
WP2 is only performed at baseline.
Study design
General study design:
This is a longitudinal study, which will be organized into three work packages (WP1,
WP2 and WP3). Enrollment will continue for an estimated period of 24 months,
depending on the time course of the epidemic curve. Last-patient-in is expected no
later than November 1st, 2023.
Patients will be referred mainly by the University Hospital of Antwerp (UZA) and
enrolled at first visit to the University Psychiatric Hospital (UPCD), where follow-up
will also take place at 3, 6 and 12 months. Patients can also be referred by the
Flemish long COVID patient support group.
Sample size:
Based on current referral rates, we expect on average 1-2 new referrals each week.
After 12 months, we expect a total of ±50 patient inclusions. In a MANOVA analysis,
a total sample size of 64 offers 80,0% power to detect a medium sized effect
(f2≥0.15). We aim for a total sample size of 130 in order to achieve higher effect
sizes and to maintain sufficient power in the subgroup analyses and in case of
missing values. As Ca is the main control group for the long COVID patient group,
these two groups are of equal size (both 50 participants). We included a smaller Cb
group to compare the immunological blood parameters between healthy volunteers
and long COVID patients.
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Study visits:
As part of routine clinical care, patients will receive psychiatric evaluation at the
University Hospital Campus UZA, and neurocognitive evaluation at Campus Duffel.
During the clinical evaluations, participants will be invited to participate in the study.
If they are interested, they will be invited to a baseline visit assessing eligibility and
covering informed consent procedure. In order to adhere to social distancing
regulations,
it
may
occur
that
this
baseline
visit
is
held
digitally
via
livestream/webcam or by telephone. In this case, patients sign their ICF digitally. To
ensure
patient
privacy,
only
in-house
fully secured internet and telephone
connections will be used for distanced baseline visits.
Once included, the participant will be invited for the baseline assessment, which
includes a broad neuropsychological examination. A blood sample will be collected
at each time point during the study.
SCREENING
BASELINE
3 MONTHS
6 MONTHS
12 MONTHS
PATIENT GROUP
Eligibility,
psychiatric
and IQ
screening
Psychiatric
assessment
Blood sample
Psychiatric
assessment
Blood
sample
Psychiatric
assessment
Blood sample
Psychiatric
assessment
Blood sample
+ NC
SUBGROUP
Neurocognitive
assessment
Neurocognitive
assessment
+ HPA
SUBGROUP
Saliva sample
Saliva sample
CONTROL
GROUP A
Eligibility,
psychiatric
and IQ
screening
Psychiatric
assessment
Blood sample
Psychiatric
assessment
Blood
sample
Psychiatric
assessment
Blood sample
Psychiatric
assessment
Blood sample
+ NC
SUBGROUP
Neurocognitive
assessment
+ HPA
SUBGROUP
Saliva sample
Screening
Baseline
1 month
2 months
3 months
CONTROL
GROUP B
Eligibility,
psychiatric
screening
and IQ
screening
Psychiatric
assessment
Blood sample
Saliva sample
Psychiatric
assessment
Blood
sample
Psychiatric
assessment
Blood sample
Psychiatric
assessment
Blood sample
Figure 5: Tasks per (sub)group for each time point of the study
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3.3 Study measurements
To begin with, possible participants will be screened for eligibility before inclusion.
First, patients with long COVID symptoms can be referred after completing the
screening tests, assessing psychological and cognitive complaints, and can be
included if these self-report questionnaires indicate neuropsychiatric symptoms. The
control
group
will
perform
a
screening
test
to
exclude
any
self-reported
psychological or cognitive complaints. If possible, these screening tests will be
organized on an online platform.
The data which will be collected for this study which are part of routine clinical
practice include the following procedures:
-
a baseline clinical psychiatric interview to determine a patient’s past and
current mental state and psychiatric diagnosis, including psychiatric rating
scales
-
a baseline neuropsychological evaluation including a standardized test
battery (optional)
-
extensive neurocognitive tests to examine and document persistent cognitive
complaints
Additionally, the following study-related procedures will be performed:
-
informed consent and eligibility screening
-
blood sample draw (max 35ml per visit)
-
saliva cortisol sampling
-
additional self-rated scales assessing insomnia, fatigue and psychopathology
-
a questionnaire regarding important bio-psycho-social factors such as familial
psychiatric antecedents, socio-economic status, social network during the
pandemic, personal experience with the COVID-related restrictions, …
3.3.1. Psychiatric interview
The psychiatric interview will be performed at baseline by the treating physician as
part of standard clinical practice and will include diagnostic evaluation using the
MINI. If the MINI was not yet performed by the treating physician, this will be
performed during the screening.
Validated self-report questionnaires will be used to assess psychopathology on each
time point of the study (see table 1).
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Table 1: Neuropsychiatric rating scales
Domain
Scale
Cut-off
SCREENING PHASE (before inclusion)
Depression,
anxiety, trauma
RMT-20
Rapid Measurement Toolkit 20 (40)
MDD ≥ 13
GAD ≥ 11
SAD ≥ 12
Panic ≥ 9
PTSD ≥ 8
Depression
BDI-21
21-item Beck depression inventory
(41)
10-18: mild
19-29:
moderate
30-63: severe
Anxiety
STAI
State-Trait Anxiety Inventory (42)
State: ≥ 39
Trauma
PCL-5
PTSD Checklist for DSM-5 (44)
≥33
IES-R
Impact
of
Event
Scale
-
Revised
(45,46)
≥33
Insomnia
ISI
Insomnia Severity Index (47)
≥10
Neurocognitive
CFQ
Cognitive
Failure
Questionnaire
(subjective) (48)
General
cut-off
score of ≥ 43.
IQ
RSPM-
SF
Raven Standard Progressive Matrices
(Short Form) (39)
IQ > 90
Substance abuse
ASSIST
Alcohol,
Smoking
and
Substances
Involvement Screening Test  (72, 73)
≥11 for alcohol,
≥4
for
other
substances
COVID-19
C19-Re
habNe
q
COVID-19
Rehabilitation
Needs
Questionnaire (70)
No cut-off
CLINICAL/EXPERIMENTAL PHASE (baseline – T1 – T2 -T3)
Diagnosis
(only
baseline)
MINI
Mini
International
neuropsychiatric
Interview (49)
Depression
HDRS-
17
17-item Hamilton Depression Rating
Scale
8-13: mild
14-18:
moderate
19-22: severe
≥23: very severe
BDI-21
21-item Beck depression inventory
(41)
Anxiety
STAI
State-Trait Anxiety Inventory (42)
Insomnia
PSQI
Pittsburgh Sleep Quality Index (50)
≥5
ISI
Insomnia Severity Index (47)
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DRFS
Dream recall frequency scale (exp)
(51)
ITI
Insomnia Type Inventory (exp)
(only if ISI >9)
Fatigue
FSS
Fatigue Severity Scale (52)
Trauma
CTQ
Childhood trauma questionnaire (53)
IES-R
Impact
of
Event
Scale
-
Revised
(45,46)
≥33
Neurocognitive
DSC
Digit Symbol Coding Subtest from
WAIS-IV
see 3.3.2.
CWI
Color Word Interference Test from the
Delis-Kaplan
Executive
Function
System
see 3.3.2.
BWT
Bourdon-Wiersma Test
DS
Digit Span Subtest from WAIS-IV
see 3.3.2.
RAVLT
Rey Auditory-Verbal Learning Test
see 3.3.2.
CFT-R
Complex Figure Test (Rey figure)
see 3.3.2.
BNT-SF
Boston Naming Test, short form
see 3.3.2.
COWA
T
Controlled
Oral
Word
Association
Test
see 3.3.2.
WFT
Word Fluency Test
see 3.3.2.
S
MR
Subtests Similarities from WAIS-IV
Subtest
Matrix
Reasoning
from
WAIS-IV
see 3.3.2.
TOL
Tower of London
see 3.3.2.
TMT
Trail
Making
Test
from
the
Delis-Kaplan
Executive
Function
System
see 3.3.2.
PPT
Purdue Pegboard Test
see 3.3.2.
Substance abuse
ASSIST
Alcohol,
Smoking
and
Substances
Involvement Screening Test  (72, 73)
COVID-19
PCFS
Post-COVID-19
Functionele
Statusschaal (71)
No cutoff
3.3.2. Neuropsychological assessment
COVID-19 patients with persistent complaints of cognitive disabilities
(more than 4 weeks post-infection) in daily life are examined with an
extensive neuropsychological test battery (see below) at the outpatient
clinic of the University Psychiatric Centre in Duffel and at the University
Hospital Antwerp. This battery covers a broad range of reliable and
valid tests divided into six cognitive domains: attention, memory,
visuospatial functions, language, executive functions and psychomotor
speed/coordination. The neuropsychological examination lasts an
average of 2 hours per participant. The tests are carried out by the
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same experienced neuropsychologist and also follow a standardized
sequence. In addition to the neuropsychological assessment, two
self-report scales are filled out by each patient. These scales are used
to detect possible complaints of fatigue and possible cognitive
problems which are experienced by the patient during daily life
activities.
A control group of on average 50 COVID-19 survivors without
persistent complaints (Ca), matched on age and years of education
with the patient group, will also undergo the neuropsychological test
battery.
●
Attention
●
The Bourdon-Wiersma Test (BWT) (54) was used to evaluate
sustained attention. The primary outcome variables are mean
rule time and total omissions.
●
The Digit Symbol Coding Subtest from the Wechsler Adult
Intelligence Scale (4th Edition) (WAIS-IV (DSC)) (58) is used to
assess divided attention and speed of information processing.
The total correct symbols and the total errors (or incorrect
symbols) within the allowed time of 120 s are used as outcome
measures.
●
The Dutch version of the Color Word Interference Test from the
Delis-Kaplan Executive Function System (D-KEFS (CWI)) (65) is
administered to measure focused attention and inhibition of
interference (card III) and alternating attention and cognitive
flexibility (card IV). The total raw score (total time) and the total
uncorrected errors for card III (evaluation of focused attention)
and card IV (evaluation of alternating attention) are used as
outcome measures
●
Memory
The Digit Span Subtest from the Wechsler Adult Intelligence
Scale (4th Edition) (WAIS-IV (DS)) (58) is used to evaluate working
memory. For the digit span forward and the digit span backward
we use the total raw score and the longest span (forward or
backward) as outcome measures.
The
Rey
Auditory-Verbal
Learning
Test (RAVLT) (54,55) is
administered to evaluate auditory-verbal memory for unrelated
information. The variables we use for the analysis are total
immediate recall (over 5 trials) (the maximum learning score is
75), long-term delayed recall (after a 20-min time interval; the
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maximum recall score is 15) and long-term delayed recognition
(after a 20-min time interval; the maximum recognition score is
15).
Visuospatial memory for related information is assessed by
means of the Complex Figure Test (Rey figure) (CFT-R) (54–56).
The two measures we use are immediate recall and long-term
delayed recall (after a 20-min time interval) of recently learned
information.
●
Visuospatial functions
The copy of the Complex Figure Test (Rey figure) (RCFT-R)
(54–56) is administered to evaluate visuospatial judgement. The
total score (with a maximum of 36) is recorded.
●
Language
In our study we administer the short form of the Boston Naming
Test (BNT-SF) (66) to measure word finding (i.e., naming of 29
objects). The outcome measure is the total correct answers (with
a maximum of 29).
●
Executive functions
The Controlled Oral Word Association Test (57) and the Word
Fluency Test (WFT) (67) are used to assess phonetic verbal
fluency and semantic verbal fluency, respectively. The total
words generated is used as outcome measure for both tests.
The
Subtests
Similarities
and
Matrix
Reasoning
from the
Wechsler Adult Intelligence Scale (4th Edition) (WAIS-IV (S) and
WAIS-IV (MR)) (58) are selected to measure verbal abstract
reasoning and non-verbal abstract reasoning, respectively. The
total raw score is used as outcome measure for both subtests.
The Tower of London Test (59) is used to evaluate planning and
problem solving. The outcome variables are the total correct
score, the total move score and the total problem-solving time.
●
Psychomotor speed/coordination
The conditions 4 and 5 of the Trail Making Test from the
Delis-Kaplan Executive Function System (D-KEFS (TMT)) (68) are
administered to evaluate psychomotor speed with a cognitive
component (cognitive flexibility) (condition 4) and psychomotor
speed without a cognitive component (condition 5). For both
conditions, the total raw score (total time) is used as outcome
measure.
The Purdue Pegboard Test (60) is used to assess psychomotor
coordination. The total raw score (or the average of three trials,
each trial with a time limit of 30 s) for the preferred hand, for the
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non-preferred hand and for both hands are used as outcome
measures.
●
Cognitive functioning in daily life
The Cognitive Failure Questionnaire (CFQ) (69) is used to detect
possible cognitive problems (or failures) which are noticed by
patients during daily life activities in the past four weeks. This
self-report scale consists of 25 items with a rating of 0 (never), 1
(rarely), 2 (sometimes), 3 (often) or 4 (very often) for each item.
The total score (with a maximum of 100) is used as outcome
measure.
●
Fatigue
The Fatigue Severity Scale (FSS) (61) is used to detect possible
complaints of fatigue in daily life by the patient. This self-report
scale consists of 9 items about fatigue, its severity and how it
can affect several daily activities. The items are scored on a
7-point rating scale from 1 (strongly disagree) to 7 (strongly
agree). The total score is used as outcome measure (with a
maximum of 63).
●
Cognitive rehabilitation
The COVID-19 patients are divided into two groups: a treatment
group and a non-treatment group. The patients in the treatment
group follow a cognitive rehabilitation program. This program
consists of 12 group sessions of one hour (one session per
week). A maximum of 6 patients participate to a group session.
Each session consists of two cognitive rehabilitation methods.
These
are
restorative therapy (which means administering
several cognitive exercises (with increased level of complexity) in
order
to
improve
the
specific
cognitive
deficits
of each
individual
patient
(as
was
detected
by
means
of
the
neuropsychological
baseline
evaluation)
with
regard
to
attention,
memory
and/or
executive
functions)
and
(2)
compensatory therapy (which means teaching compensation
strategies in order to cope better with the cognitive disabilities
concerning attention, memory and/or executive functions that
each individual patient experiences during daily life activities).
The patients in the non-treatment group receive no cognitive
rehabilitation program.
●
Neuropsychological reassessment
The
patients
in
both
groups
(treatment
group
versus
non-treatment
group)
undergo
a
neuropsychological
reassessment after approximately 3 months. For some tests
(RAVLT, CFT-T (Taylor figure), COWAT and WFT) a parallel
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version is used to minimize a possible practice effect. Of course,
for the treatment group this neuropsychological reassessment is
necessary to evaluate the progression of cognitive functioning
after 3 months of cognitive rehabilitation.
3.3.3. Blood sampling
Blood samples will be collected in a standardized manner by venipuncture. Two
blood vials will be collected and processed to perform the following analyses:
-
serum collection tube(s) allowing for HERV-W ENV and serological assays
-
plasma
collection
tubes
for
the
assessment of other immune-related
biomarkers (including but not limited to CRP, IL-6, IL-1b, TNF-a, IFN-g,
tryptophan, kynurenine, 3-OH-kynurenine, quinolinic acid, kynurenic acid,
enzymes of the kynurenine pathway).
A maximum of 35ml blood will be collected during each visit, clinical test sampling
included. Study-dedicated blood tubes will be processed for respective plasma and
serum extraction according to standardized protocols and plasma / serum will
subsequently be stored at -80°C until analysis or elimination.
Residual pellet material of whole blood centrifugation for plasma collection will be
utilized for DNA extraction to perform additional genotyping. Emerging evidence
has linked specific DNA haplotypes with increased risk of psychiatric morbidity.
Exploration of specific immune related genes in post-COVID NP patients might
reveal
insights
in
genetic
predisposition
for
infection-driven
psychiatric
decompensation. DNA sequencing of immune related loci (including but not limited
to MHC-II) will be performed on residual pellet material from centrifugated blood
collection tubes for plasma procurement if specific additional consent is obtained
from the participant. If such consent is not granted, residual pellet material will be
discarded after plasma collection.
3.3.4. Cortisol measurements (HPA axis)
Salivary cortisol measures will be used as functional measure of the HPA axis activity,
as the unbound cortisol fraction in saliva is highly correlated to plasma cortisol
(62–64). Baseline diurnal cortisol and CAR levels will be measured using saliva,
collected at home with Salivette® (Sarstedt) synthetic swabs. The rationale behind
the choice of home sampling, is to maximize normal circumstances and to minimize
stress related to the measuring procedure. Participants are also asked to perform
the sampling on two consecutive days with no anticipated excessive stress or
physical or mental efforts. The Salivette® sampling method is easy and minimally
invasive. Participants put the swabs in their mouth and move them around for
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Protocol COV-N-Psy Version 5.0_OKT2022

approximately 60 seconds. Participants are required to refrain from smoking,
drinking and eating or brushing the teeth 30 minutes before each measurement.
The sampling times are immediately upon awakening, 30, 45 and 60 minutes after
awakening (CAR). All samples will be analyzed through electrochemiluminescence
immunoassay at the laboratory of the Antwerp University Hospital. The lower
detection limit of cortisol is 0.544 ng/ml. This value is assigned whenever cortisol
levels were below the detection threshold.
3.3.5. Electronic clinical records
The following demographic and clinical variables will be collected through
electronic clinical records. If certain data is unavailable from the records, the
participant may be contacted by a member of the research team for an additional
interview.
●
Demographics: age, gender
●
Clinical diagnosis at baseline psychiatric evaluation
●
Medical history
●
Psychiatric history
●
Family psychiatric history
●
Current + past medication use
●
Metabolic parameters: weight, length, waist circumference, triglycerides (TG),
HDL-cholesterol, blood pressure, fasting plasma glucose
●
Current substance abuse: nicotine, alcohol, cannabis
●
If available, results of RT-PCR of SARS-Cov-2 on a nasopharyngeal swab or of
SARS-Cov-2 serology
●
Date of acute infection, duration of NP symptoms of long Covid
●
If available, brain imaging, polysomnography or other technical exams
performed during or after COVID-19 infection, including lab results during
acute COVID-19 infection
●
If available: inflammatory parameters in lab result during acute infection.
Otherwise: inflammatory parameters of last clinical lab result (including but
not limited to white blood cell count)
●
SARS-Cov-2 vaccination status
3.3.6. Follow-up measures
Depending
on
the
clinical
findings,
treatment
with
psychopharmacological
compounds may be initiated or patients may be enrolled in a group cognitive
remediation program contingent upon clinical indication. Follow-up visits with
psychiatric evaluation, self-rating scales and blood sampling will take place every 3
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months for a maximum of one year. Follow-up neuropsychological assessment will
take place once after 6 months.
3.4.
Endpoint measurements
“NP symptoms”
- Baseline and follow-up scores on clinical scales of depression, anxiety, and
insomnia
- (subgroup) Baseline and follow-up cognitive functioning
“Inflammation”
- Quantitative measurement of HERV-W-ENV in serum at baseline
- Calculation of systemic immune inflammatory index (SII= thrombocyte count x
neutrophile count / lymphocyte count) at baseline and follow-up
- Quantitative analysis of CRP and IL-6 at baseline and follow-up
- Quantitative analysis of kynurenine metabolites at baseline and follow-up
(subgroup) “HPA axis”
-
CAR
3.5.
Statistical analysis
CAPRI has ongoing collaborations with StatUA and BIOMINA for statistical analyses
of past and ongoing projects involving biomarker-data. In this project, we will study
cross-sectional and bidirectional longitudinal associations between NP symptoms
and Inflammation or Insomnia (cfr above).
To study the cross-sectional association using all available data, we will stack data
from all timepoints in one cross-sectional dataset. To account for the fact that one
person can have multiple observations in this combined cross-sectional dataset, we
will analyze the association between NP symptoms and inflammation / insomnia
(measured at the same timepoint) using linear mixed models with timepoint as
repeated effect and patient identifier as within-subject effect and using an
unstructured correlation matrix. Estimates from this model thus represent the
cross-sectional association between predictor and outcome.
For longitudinal analyses the effect of baseline Inflammation and Insomnia on NP
symptoms at 3- and 6-month follow-up will be evaluated using linear mixed models
with time as repeated effect and patient identifier as within-subject effect and using
an unstructured correlation matrix. Baseline predictor by time interaction (time
coded as 0, 3, or 6) will be included to test differences in slope but retained only in
models when statistically significant. Treatment group (antidepressant treatment,
insomnia treatment, cognitive remediation, a combination, or none) will be included
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as predictor. Baseline values of NP symptoms will be included as covariates, and all
other covariates measured at baseline as well.
3.6.
Management of the database
Management of the database will be performed in line with applicable regulatory
procedures by SINAPS (research unit of CAPRI and Emmaus in UPC Duffel). The
data manager of the study is Dr. Katrien Skorobogatov. The database, only including
pseudonymized (encoded) data, will be stocked on an encrypted hard drive,
accessible by the SINAPS team members. The file containing information on the
coding of the participants will be kept in a separate file, equally kept on an
encrypted hard drive, only accessible with the encryption key by the principal
investigator (Dr. Livia De Picker). The research data will be archived for a duration of
20 years after the study is completed.
3.7.
Ethical aspects
The study is submitted to the ethics committee of Antwerp University Hospital /
University of Antwerp and will be conducted in accordance with the declaration of
Helsinki. Written informed consent is necessary before inclusion and participation.
All participants will be informed about the no-fault insurance which covers any
personal damage directly or indirectly related to the study.
The participants will receive a fee of €50 for each visit to compensate for any
financial costs they may have suffered related to their participation in this study
(e.g., for transportation to and from the hospital), except for patients who
participate
in
the
neurocognitive
tests.
Instead,
patients
undergoing
the
neurocognitive tests will receive these assessments including a full report for free.
In order to clearly separate the research project from clinical treatment, which takes
place in UZA, the participants will be invited to UPCD for every time point in the
study.
All participants have the right to withdraw from the study at any moment for any
reason, since participation is strictly voluntary. The researcher has the right to
withdraw a participant for any of the following reasons:
-
Withdrawal of consent
-
Coöperation inability
-
Protocol violation
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