Self-reported health, neuropsychological tests and biomarkers in fully recovered COVID-19 patients vs patients with post-COVID cognitive symptoms: a pilot study
1Division of Clinical Neuropsychology, Corewell Health, Grand Rapids, Michigan, USA
2Department of Neurology, Corewell Health, Grand Rapids, Michigan, USA
3Department of Family Medicine, College of Human Medicine, Grand Rapids, Michigan, USA
4Department of Translational Neuroscience, College of Human Medicine, Grand Rapids, MI, USA
*Corresponding author: arnetzju@msu.eduAbstract
Objective
Substantial numbers of individuals who contract COVID-19 experience long-lasting cognitive symptoms such as brain fog. Yet research to date has not compared these patients with healthy controls with a history of laboratory-confirmed COVID-19 infection, making it difficult to understand why certain COVID patients develop post-COVID cognitive symptoms while others do not. The objective of this pilot study was to compare two groups of laboratory-confirmed post-COVID patients, with and without cognitive symptoms, on measures of cognitive and psychological functioning, self-reported perceptions of functional status and quality of life, and biomarkers of stress, inflammation, and neuroplasticity.
Methods
Using a case-control design, 17 participants were recruited from a healthcare system in western Michigan, USA in 2022 through 2024. All participants were aged 25-65 and had a positive polymerase chain reaction (PCR) test confirming previous COVID-19 infection. Ten participants reported cognitive symptoms (long COVID group) while seven were fully recovered with no residual symptoms (controls). All participants underwent an interview on their self-rated health and quality of life, a battery of neurocognitive tests, and blood draw for biomarker analysis.
Results
No group differences were detected for neuropsychological test measures except for letter fluency where the long COVID group scored significantly lower (p<.05). The long COVID group had significantly lower ratings than controls on quality of life, physical health, emotional functioning, and psychological well-being. Serum levels of nerve growth factor (NGF), a biomarker of brain plasticity, were significantly lower in the long COVID group, which was significantly more likely than controls to have serum levels of inflammatory marker (interleukin (IL)-10) values greater than or equal to the median (p=0.015).
Conclusion
Biomarker analyses suggest possible prolonged inflammatory processes in long COVID patients compared to fully recovered patients. Results of decreased neuroplastic functioning give credence to patients’ reports of post-COVID changes in brain function.
Article notes
Competing Interest Statement
The authors have declared no competing interest.
Funding Statement
Yes
Introduction
Post COVID conditions (PCC), or long COVID, is a clinical syndrome characterized by symptoms of COVID-19 that extend well beyond the initial recovery period [1]. The clinical diagnosis, termed post-acute sequela of COVID-19 (PASC), was officially defined and assigned in the International Classification of Diseases (ICD) - 10 in October of 2021. However, a widely accepted case definition and associated symptom timeframe is still under development [2]. Research suggests that approximately 30% of individuals who contract COVID-19 will go on to develop a post COVID-19 condition and experience long-lasting symptoms [3]. Although symptoms are often diverse, many patients experience persistent cognitive complaints, including poor memory and “brain fog” [1,4]. To date, four years after the onset of the COVID-19 pandemic, cognitive symptoms continue to plague a substantial proportion of individuals who have experienced the viral infection [5].
A large survey of U.S. adults conducted during a six-month period in 2021-2022 found that 46% of those with PCC reported either brain fog or impaired memory, which was associated with a decreased likelihood of working full-time [6]. Another U.S. survey of nearly 15,000 adults with prior COVID-19 infection found that 57% of individuals with PCC experienced significantly more cognitive symptoms at least daily compared to those without PCC [7]. The most commonly reported cognitive disturbances involved attention, executive functioning and memory, with brain fog or processing speed deficits reported in 40-60% of patients with long COVID [8]. Brain fog, or COVID fog [9] is often accompanied by fatigue, anxiety, and depressive symptoms similar to those seen in chronic fatigue syndrome [10]. Post-COVID cognitive symptoms are reported more frequently among individuals who had a mild initial infection [10, 11, 12] and in individuals who report prior cognitive difficulties and a diagnosis of depressive disorder [13]. Individuals with an elevated psychological symptom burden as part of their post COVID-19 clinical picture report more perceived cognitive difficulties in day-to-day life [14]. Post-COVID cognitive symptoms have also been associated with decreased quality of life and function [15].
Notably, research to date has not shown significant objective cognitive deficits in individuals with cognitive complaints related to long COVID. A study of 51 adults with post-COVID cognitive symptoms reported that they scored in the normal range on standardized neuropsychological measures. However, that study lacked a control group [14]. The results of neuropsychological tests in 53 outpatients diagnosed with COVID-19 revealed no scores in the impaired range, leading the authors to conclude that objective cognitive performance was not affected by self-reported cognitive complaints [16]. One study compared adults with post-COVID conditions (n=319) to healthy controls (n=109) and found no significant group differences in neuropsychological test results. Healthy controls in that study had not been infected with COVID-19. However, when dividing the PCC group into those with cognitive complaints (n=123) and those without (n=196), they found that those with cognitive complaints scored significantly worse on global cognition, learning, memory, processing speed, language, and executive function [15].
A number of studies [e.g.,17-23] have focused on biomarkers associated with post-COVID symptoms in an effort to better understand PCC, improve diagnosis, and develop potential therapeutic treatments. Lai et al. [21] identified interleukin (IL)-6, C-reactive protein (CRP), tumor necrosis factor alpha (TNF-alpha), and neurofilament light chain (NfL) as potential diagnostic indicators of long COVID. In a Brazilian cohort, Queiroz et al. [22] found that patients with long COVID exhibited higher levels of IL-17 and IL-2, whereas patients with no post-COVID symptoms had higher levels of IL-4, IL-6 and IL-10. While patients in that study had all been diagnosed with COVID, the authors only compared biomarkers between groups and did not report on cognitive complaints. However, due to the broad array of symptoms associated with PCC, as well as the varied study designs employed, it is difficult to draw any definitive conclusions regarding specific biomarkers. A few studies have focused more specifically on biomarkers associated with cognitive impairment [24, 25]. In a study of 710 COVID survivors, Damiano et al. [24] found no cytokines or inflammatory markers associated with cognitive performance. Vrettou et al. [25] studied neural biomarkers in a cohort of 65 long COVID patients and 29 age and sex-matched healthy controls with no known history of COVID infection. They found that levels of glial fibrillary acidic protein (GFAP), a biomarker of neural inflammation, were significantly higher among long COVID patients, but were not correlated with the presence of long COVID symptoms.
In summary, the literature to date offers sparse evidence of biomarker response to post-COVID cognitive complaints, and we lack standardized phenotyping for biomarker analyses [26]. Moreover, patients with PCC cognitive complaints do not seem to differ from healthy controls on neurocognitive testing but did score worse than PCC patients without cognitive complaints [15]. However, patients with PCC cognitive complaints may also differ in terms of psychological symptoms and self-perceived health and functional status [14, 25, 27]. In any event, large numbers of individuals with PCC continue to suffer from cognitive symptoms that do not seem to improve with time [5]. Studies to date have compared cognitive issues between COVID laboratory-positive and laboratory-negative patients [27, 28] or followed longitudinal cohorts of lab-confirmed COVID patients without comparison groups [12, 13]. The lack of studies involving controls with a history of laboratory-confirmed COVID-19 infection makes it difficult to enhance our understanding of why certain COVID patients develop PCC cognitive symptoms while others do not. To address these gaps in the literature, the aim of the present pilot study was to compare two groups of laboratory confirmed post-COVID patients, with and without cognitive symptoms, on measures of cognitive and psychological functioning, self-reported perceptions of functional status and quality of life, and biomarkers of stress, inflammation, and neuroplasticity.
Materials and Methods
Study design
This was a pilot study utilizing a case-control design.
Study participants
The sample was comprised of a total of 17 participants who had previously contracted COVID-19. All were recruited from Corewell Health medical facilities in western Michigan, U.S. Inclusion criteria included being between the ages of 25-65, at least 6 months after COVID infection, and a positive polymerase chain reaction (PCR) test confirming COVID infection. All participants were placed into one of two groups, a symptomatic group based upon self-report of cognitive symptoms (“long COVID”), or an asymptomatic control group. The asymptomatic group was fully recovered from COVID, with no residual symptoms. Attempts were made to age and sex match the symptomatic and control group.
Data collection
Recruitment took place between March 4, 2022 and April 10, 2024. Each participant met with a study coordinator who verbally explained the nature of the study, the research intent, and risks. Participants then read and signed informed consent forms prior to participation. All participants underwent an interview on their self-rated health and quality of life, a battery of neurocognitive tests, and blood draw for biomarker analysis. Each participant was reimbursed $75 upon completion of the approximately 2 - hour evaluation.
Self-report symptoms
All participants completed a brief structured interview assessing symptom complex and time frame/course. The self-report measures focused specifically on mood, emotional functioning and quality of life. Mood was measured using the Beck Depression Inventory [29] and Beck Anxiety Inventory [30].
Emotional functioning and quality of life were measured using the Short Form Health Survey SF-36 [31; 32] and the EuroQuol 5-Dimension EQ-5D [33]. Descriptions of each measure are provided in Table 1.
Neurocognitive testing
The interview was followed by a 90-minute neurocognitive battery assessing various aspects of cognition including estimated premorbid functioning, attention, processing speed, verbal fluency, learning and memory, visual planning, and executive functioning (e.g., problem-solving, multitasking, sustained concentration). An overview and description of the tests is provided in Table 2. All measures used in the current study were well known for their strong psychometric properties.
Biomarker analysis
Blood serum and plasma specimens were taken for biomarker analysis. All blood sampling was conducted at the health system laboratory. For each participant, 30 mL blood was drawn by a licensed phlebotomist, and the time of day of sample collection was recorded. All samples were frozen and stored in a –80 8C freezer for later analysis. Analysis focused on biomarkers of stress (cortisol), anti-stress or recovery (dehydroepiandrosterone sulphate, DHEA-S), inflammation (IL-6, IL-10), neuroplasticity (brain-derived neurotrophic factor, BDNF, and nerve growth factor, NGF) and neurodegenerative disease (neurofilament light chain, NfL). Analyte concentrations in both serum and plasma samples were measured using human-specific 96 well ELISAs with the exception of NfL, which was analyzed by Single Molecule Array (SIMOA) technology. The following kits were used to generate data: Cortisol (Boster #EK7002, Pleasanton, CA; sensitivity > 20 ng/ml), DHEA-S (Invitrogen #EIAD HEA, Carlsbad, CA; > 90.9 ng/ml), IL-6 (BioLegend #430507, San Diego, CA; > 1.6 pg/ml), IL-10 (BioLegend #430607, San Diego, CA; > 2 pg/ml), BDNF (Origene #EA100205, Rockville, MD; > 2 pg/ml), NGF (AVIVA #OKEH00186, San Diego, CA; > 15.6 pg/ml), and NfL (Quanterix #193186, Billerica, MA; > 1.38 pg/ml). Manufacturer’s instructions were followed for all assays. Duplicate samples were quantified using standard curves based on calibrators of known concentration; intra-assay % CVs ≤ 5.2% for all assays.
Data analysis
Statistical analysis was conducted using SPSS Statistics version 29 (IBM Corp, Armonk, NY), with a two-sided p value <.05 deemed statistically significant. Due to the small sample size, all comparisons between the long COVID and asymptomatic control groups on self-report measures and neurocognitive tests were conducted using non-parametric tests, specifically, Chi square or Fishers’ exact tests for discrete variables and Mann-Whitney U-tests for continuous variables. Group comparisons for biomarker values utilized the natural log (Ln) for all biomarkers and were conducted using independent samples T-tests. Since the study is based on relatively few participants and there are no published data on recommended cut-off levels for biomarkers applicable to PCC patients, additional analyses were conducted by dichotomizing the biomarkers into below the median vs equal to or above the median based on aggregate data for all participants. Using these cutoffs, group comparisons were conducted to examine whether the proportion of participants across the two groups above and below the median differed statistically. In a final step, a pro-inflammatory index was created by summing up the absolute values for IL-10 and IL6. The long COVID scores were compared with the controls using total scores as well as the Ln transformed scores. The serum pro-inflammatory Index was dichotomized into less than the median (=5.4005 pg/mL) vs greater than or equal to the median. Fisher’s exact 2-sided test was used for these analyses, with significance set at p<.05.
Ethical Considerations
The study was approved by the Institutional Review Board at Corewell Health (IRB nr. 2020-601).
Results
Demographic characteristics of study participants are summarized and compared by group in Table 3. The mean age was 42 years in the long COVID group and 44.32 in the controls. All but one participant was female, all but two identified as White, and none identified as Hispanic. Most participants in both groups had a high school diploma or associate degree as their highest educational degree. All seven control group participants were employed, compared to 6 of 10 in the long COVID group. Three of the 4 unemployed reported that their unemployment was due to their post COVID condition. None of the group differences were statistically significant.
Self-report measures and neurocognitive tests
Group differences on self-report measures for emotional functioning/quality of life and mood, and on objective neuropsychological tests are summarized in Table 4. On self-report measures, individuals with long COVID scored significantly higher than controls, indicating a higher level of problems on the quality of life measures for usual activity (2.20 vs. 1.00, p=.002) and pain/discomfort (2.20 vs. 1.17, p=.007), as well as on the EuroQol 5-Dimension (EQ-5D EQ5D3L) total score (9.50 vs. 5.50, p<.001). There were no significant group differences for mobility, self-care, and anxiety/depression. The long COVID group scored significantly lower (mean 38.10) than the non-symptomatic group (mean 89.17) on the visual analogue scale (VAS) for self-rated health, ranging from 0 (worst health you can imagine) to 100 (best health you can imagine). On the SF-36 quality of life measures, the long COVID group scored significantly lower than the healthy controls on all eight dimensions (p<.01). Thus, those with post-COVID cognitive complaints experienced greater limitations in physical and social activities due to physical and emotional health problems, and reported worse bodily pain, general mental health, and lower vitality, i.e., less energy and more fatigue, than controls. The long COVID group also scored significantly higher on both mood measures (depression, mean 27.50 vs. 3.86, p<.001; anxiety, 21.40 vs. 4.57, p=.002) than healthy controls.
With regard to objective neuropsychological test measures, there were no statistically significant group differences detected for premorbid functioning, learning and memory, attention, processing speed, executive functioning, or visual planning. By contrast, the long COVID group scored significantly lower on two measures of language related to letter fluency (raw score, 26.80 vs. 38.00, p=.043; T-Score 34.10 vs. 46.71, p=.019, Table 4).
Blood Biomarkers
Comparison of mean biomarker values between groups is presented in Table 5. The only significant difference between groups was for serum NGF levels, which was significantly lower in the long COVID group (mean 9.72) compared to the control group (13.52, p= .038).
When comparing groups based on biomarker scores below and above the median, the long COVID group was significantly more likely than controls to have serum IL-10 values ≥ the median (p=0.015). Eight out of ten participants in the long COVID group were classified into the ≥ median group vs 1 of 7 in the control group. The increased inflammatory drive in long COVID patients was confirmed in terms of the combined pro-inflammatory index, which included IL-6 as well as IL-10. Nine out of 10 patients in the long COVID group exhibited serum pro-inflammatory index values ≥ the median compared to one out of seven in the control group (Fisher’s exact test, p = 0.042). For all other biomarkers, there were no significant differences in terms of distribution of variables into below vs equal to and above the median.
Discussion
This exploratory pilot study compared two groups of laboratory-confirmed post-COVID patients at least 6 months after having been diagnosed with laboratory-confirmed COVID-19. One group was comprised of individuals who had fully recovered (controls) and the other those continuing to experience cognitive difficulties, referred to here as the long COVID group. To the best of our knowledge, this study of cognitive symptoms was the first to use a study design where both the PCC (long COVID) and control groups had a history of PCR-verified COVID-19 infection. Previous studies on cognitive impairment defined healthy controls as individuals who had not had laboratory-confirmed COVID [7, 15, 25, 28]. In an effort to understand why long COVID patients experience prolonged cognitive difficulties such as brain fog, the current study aimed to examine and compare self-reported and objective measures in PCC patients with cognitive complaints with fully recovered COVID patients. The overall study objective was to inform the current understanding of neuropathophysiological disease mechanisms contributing to non-recovery from COVID-19. Moreover, we were interested in findings that might inform the clinical management of PCC.
From a cognitive standpoint, no significant differences were seen between the two groups as it pertained to educational attainment or reading ability which is the most commonly used premorbid estimate for neuropsychological purposes. Given comparable premorbid estimates, with the exception of the letter fluency scores on the Controlled Oral Word Association Test (COWAT), this study found no significant differences between groups in the neurocognitive test battery. This is in line with previous research that found normal cognitive test results in adults with post-COVID cognitive symptoms [14, 16]. By contrast, when dividing the PCC group into those with and without cognitive complaints, Ariza et al. [15] found that those with cognitive complaints scored significantly worse on global cognition, learning, memory, processing speed, language, and executive function. However, although our findings were isolated to COWAT, this finding per se is novel since it has never been reported before, and the finding supports PCC patients’ self-reports of cognitive inefficiency and perhaps COVID fog. Specifically, this task measures one’s ability to engage in higher level thinking (a language task) under time constraint. This could help to explain patients’ reports of being able to cognitively engage but feeling that they are foggy and that things take longer and do not seem as automatic.
More consistent group differences were found on self-report measures assessing functional status, quality of life and mood. Specifically, the long COVID group in this study scored significantly and consistently worse than controls on most validated self-report measures, indicating lower ratings on quality of life, physical health, emotional functioning, and psychological well-being. Ariza et al. [15] also reported lower quality of life and functioning scores among post-COVID patients compared to healthy controls. However, unlike the present study that focused only on PCC patients with cognitive complaints, the study by Ariza et al. [15] encompassed patients with a variety of post-COVID complaints. Moreover, the control group in that study had not had a COVID-19 infection. By contrast, controls in the current study were fully recovered COVID-19-infected patients who had not experienced any post-COVID symptoms.
With regard to blood biomarkers, the current study presents intriguing evidence in terms of decreased neuroplastic functioning in PCC patients vs the control group, as reflected in decreased serum levels of nerve growth factor (NGF). NGF is a neurotrophic protein that helps regulate neuronal and neurite growth [42] as well as neuronal phenotypic maintenance and immune function [43]. Brain plasticity and neuro-immunological functioning are closely related. Our findings suggest a sustained attenuation of brain neuroplastic activity. Such findings might contribute to the numerous patient-perceived reports of attenuated neurocognitive functioning, although possibly not severe enough to be reflected in a systematic decrease in objective neuropsychiatric test scores. Of note, self-rated health is a much better predictor of future health and morbidity status than any clinical sign or laboratory test [44, 45]. It might be that the body’s internal sensor system is more sensitive and advanced than our current arsenal of clinical and laboratory testing.
However, when comparing groups based on biomarker scores below and above the median, the long COVID group was significantly more likely than controls to have serum values ≥ the median for both IL-10 alone as well as for the inflammatory index of IL-6 and IL-10 combined. Lai et al. [21] identified interleukin (IL)-6 as a potential indicator of long COVID, while Queiroz et al. [22] found that patients with no post-COVID symptoms had higher levels of IL-4, IL-6 and IL-10. However, neither of those studies focused specifically on long COVID patients with cognitive complaints. Two studies that did study long COVID patients with cognitive complaints found no cytokines or inflammatory markers associated with cognitive performance [24,25]. Another common limitation in the above cited work is the lack of objective verification of COVID-19 infection history. This limits the ability to determine whether findings are related to COVID-19 infection history specifically or due to other possible confounders.
When inflammatory marker scores were grouped above and below the median, a larger proportion of long COVID patients fell into the ≥ median group for IL-10. IL-10 represents both a pro- and anti-inflammatory marker [46]. In the current study, we used it as a pro-inflammatory marker. Prior research has also suggested that IL-10 is related to self-reported energy [47]. In the latter case, our findings might suggest a compensatory mechanism by which the body is trying to increase energy to counter the low energy and high fatigue symptoms reported by a majority of long COVID patients. These findings of ≥ median levels of IL-6 and IL-10 support the hypothesis that there is residual activation of the inflammatory systems in the long COVID group versus those that have recovered fully from PCR-verified COVID-19. Looking at the proportion of participants falling into the high vs. low serum pro-inflammatory group, all except one person in the long COVID group fell into the high serum pro-inflammatory group vs one out of seven in the control group. Thus, overall, using several different definitions of pro-inflammation, the long COVID group systematically showed a pattern of heightened inflammation as compared to the control group that has fully recovered from their COVID-19 infection.
Limitations
These results should be viewed in consideration of several study limitations. First and foremost is the small sample size, which makes generalizability to other populations difficult, although we used a rigorous assessment scheme. Small sample sizes have reduced statistical power to detect true effects and results may be affected by outliers. However, we saw a consistent pattern of lower scores on all self-rated health and quality of life measures, and higher depression and anxiety scores in the long-COVID group, which serves as validation for the group differences seen on biomarkers when examining values above and below median scores. The study sample was not diverse, as participants were primarily female and White. Notably, this was an exploratory pilot study that was, to the best of our knowledge, unique in the comparison of COVID survivors with and without post-illness cognitive symptoms. Thus, our control group had lab-verified histories of COVID infection from which they had fully recovered.
Implications for clinical care
Results of this pilot study point to few but possibly clinically important differences between long COVID and control patients with regard to neurocognitive tests, but substantial differences related to physical and emotional functioning and quality of life. Although this was a small sample, results also suggest possible prolonged inflammation in the long COVID group, which has been suggested previously [27], however, without using a proper reference group. An interesting observation is the parallel between this disorder and chronic fatigue syndrome (CFS), as both have patients reporting mood issues such as anxiety [48]. This raises the question of what we can learn from the experience of patients with CFS and whether that can be applied to long COVID patients with cognitive complaints. This may include consideration of the types of clinical teams necessary to support the patients and their caregivers as well as treatment. For example, there is no standard of care when it comes to treatment for CFS. Anhydrous Enol-Oxaloacetate, (AEO) a nutritional supplement, has been anecdotally reported to relieve physical and mental fatigue and is diminished in CFS patients [49]. Could this be a direction for us to consider for quality-of-life outcomes for long COVID patients as well? Another factor to consider is the resources needed to create longitudinal and multidisciplinary interventions that can support patients long-term - encompassing perceptions of quality of life, physical functioning, emotional health and wellbeing and vocational rehabilitation - and the cost associated with such efforts. Combined exercise and behavioral support, developed with extensive patient and stakeholder engagement, is being tested in a randomized controlled trial in the United Kingdom [50]. Should it prove to be clinically cost-effective for people with long-COVID, there is an opportunity to create similar programs.
In addition to the above, our results also support the importance of considering neuroinflammatory processes as treatment to this point has mostly focused on behavioral and cognitive interventions, assuming there are no residual neuro-inflammatory processes or deficits. This points to the importance of applying a multidisciplinary approach in addressing long COVID patients, including a rigorous assessment to detect possible residual systemic inflammation and reduced neuroplasticity. Such an approach could help to identify which patients may transition to experiencing long COVID and is likely to expand the arsenal of choices of promising and evidence-based treatment strategies with the ultimate aim to design rigorous clinical trials.
Conclusions
Long COVID patients with cognitive complaints experience significantly more anxiety and depression, lower self-rated health, and lower physical and emotional functioning and quality of life compared to fully recovered COVID-19 patients, although only one neurocognitive test differed between groups. Biomarker analyses suggest possible prolonged inflammatory processes in long COVID patients.
Moreover, results of decreased neuroplastic functioning give credence to patients’ reports of changes in brain function. Future studies in larger, more diverse samples are required to fully understand these differences and to develop effective clinical treatments for those with cognitive difficulties.
Data Availability
All relevant data are within the manuscript and its Supporting Information files.
Acknowledgements
The authors extend their gratitude to the patients who participated in the study and to John Beck, BS for conducting the biomarker analysis. This study was financially supported by a grant from the Spectrum Health Foundation (Grant nr. FDN 35911-2020-601).
Supporting Information
S1 Dataset (XLSX)