key: cord-0302037-2mssofo7
authors: Peluso, M. J.; Sans, H. M.; Forman, C. A.; Nylander, A. N.; Ho, H.-e.; Lu, S.; Goldberg, S. A.; Hoh, R.; Tai, V.; Munter, S. E.; Chenna, A.; Yee, B. C.; Winslow, J. W.; Petropoulos, C. J.; Martin, J. N.; Kelly, J. D.; Durstenfeld, M. S.; Hsue, P. Y.; Hunt, P. W.; Greene, M.; Chow, F. C.; Hellmuth, J.; Henrich, T. J.; Glidden, D. V.; Deeks, S. G.
title: Plasma markers of neurologic injury and systemic inflammation in individuals with self-reported neurologic post-acute sequelae of SARS-CoV-2 infection (PASC)
date: 2021-11-04
journal: nan
DOI: 10.1101/2021.11.02.21265778
sha: 2c0071f3df140f7cf17a7464d5091937a51d4db3
doc_id: 302037
cord_uid: 2mssofo7

Background: The biologic mechanisms underlying neurologic post-acute-sequelae of SARS-CoV-2 infection (PASC) are incompletely understood. Methods: We measured markers of neuronal injury (glial fibrillary acidic protein [GFAP], neurofilament light chain [NfL]) and soluble markers of inflammation among a cohort of people with prior confirmed SARS-CoV-2 infection at early and late recovery following the initial illness (defined as less than and greater than 90 days, respectively). The primary clinical outcome was the presence of self-reported central nervous system (CNS) PASC symptoms during the late recovery timepoint. We compared fold-changes in marker values between those with and without CNS PASC symptoms using linear mixed effects models and examined relationships between neurologic and immunologic markers using rank linear correlations. Results: Of 121 individuals, 52 reported CNS PASC symptoms. During early recovery, those who went on to report CNS PASC symptoms had elevations in GFAP (1.3-fold higher mean ratio, 95% CI 1.04-1.63, p=0.02), but not NfL (1.06-fold higher mean ratio, 95% CI 0.89-1.26, p=0.54). During late recovery, neither GFAP nor NfL levels were elevated among those with CNS PASC symptoms. Although absolute levels of NfL did not differ, those who reported CNS PASC symptoms demonstrated a stronger downward trend over time in comparison to those who did not report CNS PASC symptoms (p=0.041). Those who went on to report CNS PASC also exhibited elevations in IL-6 (48% higher during early recovery and 38% higher during late recovery), MCP-1 (19% higher during early recovery), and TNF-alpha (19% higher during early recovery and 13% higher during late recovery). GFAP and NfL correlated with levels of several immune activation markers during early recovery; these correlations were attenuated during late recovery. Conclusions: Self-reported neurologic symptoms present >90 days following SARS-CoV-2 infection are associated with elevations in markers of neurologic injury and inflammation at early recovery timepoints, suggesting that early injury can result in long-term disease. The correlation of GFAP and NfL with markers of systemic immune activation suggests one possible mechanism that might contribute to these symptoms. Additional work is needed to better characterize these processes and to identify interventions to prevent or treat this condition.

1.04-1.63, p=0.02), but not NfL (1.06-fold higher mean ratio, 95% CI 0.89-1.26, p=0.54). During late 66 recovery, neither GFAP nor NfL levels were elevated among those with CNS PASC symptoms. 67 Although absolute levels of NfL did not differ, those who reported CNS PASC symptoms 68 demonstrated a stronger downward trend over time in comparison to those who did not report CNS 69 PASC symptoms (p=0.041). Those who went on to report CNS PASC also exhibited elevations in IL-6 70 (48% higher during early recovery and 38% higher during late recovery), MCP-1 (19% higher during 71 early recovery), and TNF-alpha (19% higher during early recovery and 13% higher during late 72 recovery). GFAP and NfL correlated with levels of several immune activation markers during early 73 recovery; these correlations were attenuated during late recovery. 74 75 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted November 4, 2021. ; https://doi.org/10.1101/2021.11.02.21265778 doi: medRxiv preprint Conclusions: Self-reported neurologic symptoms present >90 days following SARS-CoV-2 infection 76 are associated with elevations in markers of neurologic injury and inflammation at early recovery 77 timepoints, suggesting that early injury can result in long-term disease. The correlation of GFAP and 78 NfL with markers of systemic immune activation suggests one possible mechanism that might 79 contribute to these symptoms. Additional work is needed to better characterize these processes and 80 to identify interventions to prevent or treat this condition.

. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted November 4, 2021. ; https://doi.org/10.1101/2021.11.02.21265778 doi: medRxiv preprint BACKGROUND 82 There is an urgent need to understand the pathophysiology that underlies the post-acute sequelae of 83 SARS-CoV-2 infection (PASC), a condition characterized by persistent symptoms in some individuals 84 recovering from coronavirus disease 2019 (COVID-19). 1 While a spectrum of symptoms is reported 85 among individuals experiencing PASC, neurologic symptoms are particularly common. [1] [2] [3] [4] [5] Limited 86 data are available on the biologic predictors and correlates of these symptoms. 87 Neurologic involvement during COVID-19 is common. 6-8 Acute illness is associated with substantial 88 immune activation 9-11 and central nervous system (CNS) dysfunction. 11 In a prior analysis utilizing a broad case definition of PASC (i.e., presence of any 1 of 32 COVID-19-03 attributed symptoms), we found that differences in levels of inflammatory markers predicted the 04 presence of symptoms >90 days following COVID-19. 38 In the current report, we investigated a more 05 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted November 4, 2021. ; https://doi.org/10.1101/2021.11.02.21265778 doi: medRxiv preprint specific outcome defined by 5 self-reported neurologic symptoms. We evaluated markers of CNS 06 injury and systemic inflammation among those with and without this more specific phenotype. A better 07 understanding of the relationships between these markers among individuals with neurologic 08 manifestations of PASC could help in identifying therapies to prevent and/or manage this condition as 09 the pandemic continues. Coronavirus (LIINC) cohort; NCT04362150). 41 The vast majority (78%) had not been hospitalized 16 during the acute phase. The determination of eligibility was agnostic to the presence or absence of 17 persistent SARS-CoV-2 attributed symptoms. 18 A research coordinator administered a study questionnaire at early (=<90 days) and late (>90 days) 19 recovery time points following COVID-19 symptom onset. Participants were queried regarding the 20 presence of 32 symptoms, including 8 neurologic symptoms (Supplemental Table 1 CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

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The primary clinical outcome was central nervous system (CNS) PASC, defined as the presence of at 29 least one CNS symptom at a late recovery visit occurring >90 days from initial COVID-19 symptom 30 onset. These symptoms included: memory/concentration issues, headache, vision problems, 31 dizziness, and balance issues. We selected these symptoms because they were felt to best reflect 32 dysfunction of the central nervous system and most likely to associate with biologic processes that 33 could be identified using the two primary biomarker outcomes. A secondary analysis examined any 34 neurological symptom, which included the following in addition to the central neurological symptoms: 35 problems with smell or taste, smelling an odor that is not really present, and numbness/tingling 36 (Supplemental Table 2 ). 37 Biomarker assays 38 Plasma biomarker measurements were performed using the fully automated HD-X Simoa platform at 39 two timepoints: early recovery (median 52 days) and late recovery (median 123 days). Those 40 performing the assays were blinded to clinical information. The primary analytes were plasma GFAP 41 and NfL measured using the GFAP Discovery and NF-light Advantage kit assays, respectively 42 (Quanterix). We also measured levels of markers that have been found to be important during acute 43 SARS-CoV-2 infection 10,11 using multiplex (Cytokine 3-PlexA: IL-6, IL-10, TNF-alpha) or single-plex 44 (IFNγ, IP-10, MCP-1) kits. SARS-CoV-2 receptor binding domain (RBD) IgG was also assayed. All 45 assays were performed according to the manufacturer's instructions and assay performance was 46 consistent with the manufacturer's specifications. 48 We log-transformed all biomarkers to reduce influence of outliers and to permit interpretation of fold-49 changes. As in prior work, 38 we compared the ratio of the mean transformed values for each 50 biomarker between those with and without persistent neurologic symptoms using linear mixed effects 51 models with terms for PASC, time period (early versus late recovery), and their interaction. This 52 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted November 4, 2021. ; https://doi.org/10.1101/2021.11.02.21265778 doi: medRxiv preprint approach permits comparison of the values at early and late time points as well as assessment of 53 whether trajectories in marker values differ between those with and without persistent symptoms. We 54 calculated fold-changes and 95% confidence intervals by exponentiating the coefficients to give the 55 ratio between the untransformed biomarker values. We used Spearman correlations to evaluate 56 relationships between levels of neurologic and immune markers. 

Study participants 65 The study included 121 individuals with primary outcome data ( Table 1) CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted November 4, 2021. ; https://doi.org/10.1101/2021.11.02.21265778 doi: medRxiv preprint Fifty-two individuals, of whom the majority were women, reported CNS symptoms at the late recovery 74 timepoint (Table 1) 83 We first compared levels of each marker measured during early recovery between those who went on 84 to report CNS PASC symptoms and those who did not ( Figure 1 , Supplemental Table 3 ). At the early 85 recovery timepoint, those who went on to report CNS PASC had significantly higher levels of GFAP 86 (1.3-fold higher mean ratio, 95% CI 1.04-1.63, p=0.02), but not NfL (1.06-fold higher mean ratio, 95% 87 CI 0.89-1.26, p=0.54). Those who went on to report CNS PASC also had higher levels of IL-6 (1.48-88 fold higher mean ratio, 95% CI 1.12-1.96, p=0.006), MCP-1 (1.19-fold higher mean ratio, 95% CI 89 1.01-1.40, p=0.034), and TNF-alpha (1.19-fold higher mean ratio, 95% CI 1.06-1.34, p=0.003) 90 compared to those who did not report CNS PASC. Trends for other markers were in a similar 91 direction, although the differences did not achieve statistical significance. 92 We next compared levels of each biomarker measured during late recovery between those with and 93 without self-reported CNS PASC at this visit ( Figure 1 , Supplemental Table 3 ). No significant 94 differences were detected in GFAP or NfL between those with and without PASC ( Figure 1 ). Those 95 reporting persistent CNS PASC symptoms had persistent elevations in IL-6 (1.38-fold higher mean 96 ratio, 95% CI 1.07-1.77, p=0.013), and TNF-alpha (1.13-fold higher mean ratio, 95% CI 1.02-1.26, 97 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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p=0.022). Interferon-gamma was lower (0.71-fold difference, 95% CI 0.55-0.91, p=0.007). Levels of 98 SARS-CoV-2 RBD IgG did not differ between groups at either the early or late timepoints. 99 Changes in levels of biomarkers over time 00 To examine changes in the levels of these markers between the early and late recovery timepoints, 01 we used mixed models to indicate changes over time among those with and without CNS PASC 02 symptoms ( Figure 1 , Supplemental Table 3 ). Significant differences in trends of NFL (p=0.041), MCP-03 1 (p=0.019), and IFN-gamma (p=0.012) were noted between the CNS PASC and non-CNS PASC 04 groups. As predicted from the cross-sectional analyses, consistently higher levels of IL-6 and TNF-05 alpha were observed, although the trends in the levels of these markers did not differ between 06 groups. 07 Relationships between neurologic and inflammatory markers 08 To examine relationships between the neurological markers and markers of inflammation, we 09 performed nonparametric pairwise analyses at early and late recovery timepoints ( Figure 2 ). GFAP 10 levels weakly correlated with MCP-1 (r=0.21, p=0.02) and IL-6 (r=0.18, p=0.054) at the early 11 timepoint and with IL-6 at the late timepoint (r=0.19, p=0.043). NfL correlated with MCP-1 (r=0.41, 12 p<0.001), IL-6 (r=0.23, p=0.012), IFN-gamma (r=0.28, p=0.003), and TNF-alpha (r=0.32, p<0.001) at 13 the early timepoint and with MCP-1 (r=0.31, p<0.001) at the late timepoint. In addition, there was a 14 strong correlation between NfL and SARS-CoV-2 IgG at the early timepoint (r=0.40, p<0.001). 15 Influence of symptoms during acute infection 16 We did not identify significant differences in levels of markers at either recovery timepoint between 17 those with and without prior CNS symptoms during acute infection (Supplemental Table 4 ). For some 18 markers, we noted non-significant trends toward differential changes over time in groups with and 19 without CNS symptoms during acute infection. These included NfL (more steep decline among those 20 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted November 4, 2021. ; https://doi.org/10.1101/2021.11.02.21265778 doi: medRxiv preprint with acute CNS symptoms, p=0.066) and anti-RBD IgG (less steep decline among those with acute 21 CNS symptoms, p=0.063). 23 Because of the relationship between age and levels of NfL, 42 we performed an age-adjusted analysis 24 which did not change the primary results (no new relationship between NfL and PASC was identified 25 (Supplemental Table 5 ). We also repeated the primary analysis adjusting for age, sex, and prior 26 hospitalization status which did not change the interpretation of the results (Supplemental Table 6 ), 27 although some of the relationships were slightly attenuated. 28 We performed a secondary analysis in which individuals reporting any symptom that could be 29 attributed to a primary neurologic cause (including the peripheral nervous system) were compared 30 against individuals reporting no neurologic symptoms (Supplemental Table 7 ). In this analysis, the 31 elevation in GFAP seen at early follow-up among those reporting any neurologic PASC symptom was 32 slightly attenuated (mean ratio 1.24, 95% CI 1.00-1.55, p=0.052). Similar elevations were seen in IL-33 6, MCP-1, and TNF-alpha at early follow-up among those reporting any neurologic PASC symptom. 34 At late follow-up, those with any neurologic PASC had higher levels of IL-6, and TNF-alpha. The 35 difference in interferon-gamma seen among those with CNS PASC in the primary analysis was no 36 longer statistically significant. 37 Finally, because we have previously found that PASC is associated with elevations in certain 38 markers, we performed an analysis comparing those with CNS PASC to those reporting no symptoms 39 of any kind during late recovery (Supplemental Table 8 ). The interpretation of the primary results was 40 again unchanged, although some of the findings were attenuated. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted November 4, 2021. ; https://doi.org/10.1101/2021.11.02.21265778 doi: medRxiv preprint A large proportion of individuals with PASC experience symptoms that may be attributed to nervous 44 system dysfunction, [1] [2] [3] [4] [5] CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted November 4, 2021. ; https://doi.org/10.1101/2021.11.02.21265778 doi: medRxiv preprint on to experience CNS PASC during late recovery appears to be more complex than identifying those 92 who report neurologic symptoms during the acute phase of illness. Our analysis has several important limitations. First, while recruitment was agnostic to the presence 07 of persistent symptoms, the cohort is not representative of the general population with PASC. 08 Second, we relied on self-report to ascertain the presence of symptoms. This risks misattribution of 09 symptoms to neurologic causes and therefore misclassification of individuals as having CNS PASC. 10 In addition, it is difficult to disentangle neurologic symptoms from non-neurologic symptoms which 11 might co-occur and it is possible that differences are driven by more severe PASC in general rather 12 than neurologic symptoms specifically. Affective symptoms may also co-occur and be inter-related. 13 Third, we did not include any objective neurologic measurements, and studies that do include such 14 measurements (which may include detailed neurological history and examination, neurocognitive and 15 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted November 4, 2021. ; https://doi.org/10.1101/2021.11.02.21265778 doi: medRxiv preprint neuropsychiatric testing, and/or neuroimaging) are likely to be more informative. Fourth, we 16 measured a limited set of biomarkers. Our measurements were all taken in blood, and while there are 17 established relationships between blood and CSF measurements in other disease conditions, 62,63 18 these have yet to be established for COVID-19. 28 For this reason, more detailed studies that include 19 CSF analyses will be critical. Finally, paired pre-pandemic specimens were not available, and it is CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted November 4, 2021. ; https://doi.org/10.1101/2021.11.02.21265778 doi: medRxiv preprint microvascular brain pathology by cleaving NEMO in brain endothelial cells. Nat Neurosci. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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Total PASC symptoms, mean (range) median (IQR) 3.3 (0 -18) 1 (0 -6) 6.9 (1 -18) 6.5 (4 -9) 0.64 (0 -7) 0 (0 -1)

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The copyright holder for this preprint this version posted November 4, 2021. Fainting spells 0 (0) 0 (0) 0 (0) Pain in your arms, legs or joints such as knees and hips 9 (7.4) 9 (17.3) 0 (0) . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted November 4, 2021. ; https://doi.org/10.1101/2021.11.02.21265778 doi: medRxiv preprint Back pain 7 (5.8) 6 (11.5) 1 (1.5)

Trouble sleeping 32 (26.5) 26 (500) 6 (8.7)

Menstrual cramps or other problems with your periods 2 (1.7) 2 (3.9) 0 (0) Pain or problems during sexual intercourse 0 (0) 0 (0) 0 (0)

All values listed are number (percentage) unless otherwise specified. IQR = interquartile range.

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The copyright holder for this preprint this version posted November 4, 2021. P-values reflect group comparisons during early and late recovery, as well as comparison of change over time between groups. GFAP, glial fibrillary acidic protein; NfL, neurofilament light chain; IL-6, interleukin-6; TNF-alpha, tumor necrosis factor alpha; IL-10, interleukin-10; IFN-gamma, interferon-gamma; MCP-1, monocyte chemoattractant protein 1; IP-10, interferon-gamma induced protein 10; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; IgG immunoglobulin G. Early recovery represents a median of 52 days post-SARS-CoV-2 symptom onset (or positive PCR); late recovery represents a median of 123 days post-SARS-COV-2 symptom onset (or positive PCR).

Data reflect nonparametric correlations between markers at early and late recovery timepoints. GFAP, glial fibrillary acidic protein; NfL, neurofilament light chain; IL-6, interleukin-6; TNF-alpha, tumor necrosis factor alpha; IL-10, interleukin-10; IFN-gamma, interferon-gamma; MCP-1, monocyte chemoattractant protein 1; IP-10, interferon-gamma induced protein 10; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; IgG immunoglobulin G. Early recovery represents a median of 52 days post-SARS-CoV-2 symptom onset (or positive PCR); late recovery represents a median of 123 days post-SARS-COV-2 symptom onset (or positive PCR).

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