SARS-CoV-2 is able to infect different tissues once it binds to Angiotensin-converting enzyme 2 (ACE2) receptor present in several organs[20]. There is strong evidence that SARS-CoV-2 infection evolves extrapulmonary systems, leading to multisystemic complications during and after the acute infection[21]. The early involvement of the nervous system is well documented.[1, 13] The neurological manifestations include encephalopathy, Guillain-Barré syndrome, acute disseminated encephalomyelitis, ischemic stroke, epilepsy and neuropathy as part of a broad spectrum that continues to be described.[13, 22–24] For a better understanding of how COVID-19 can impact nervous system and to validate candidate biomarkers for a practical approach of severity assessment, we enrolled 108 subjects from a 706-patient population admitted at the HUCFF ICU during the pandemics, between July 2020 and October 2021.
In our study, it was possible to observe that all investigated biomarkers (GFAP, NfL, TAU, UCH-L1) presented significantly higher levels among severe COVID-19 when compared to health controls (p < 0.01) in peripheral blood. However, there was no statistical significance between the same biomarkers when mild COVID-19 and health control groups were compared. Among COVID-19 positive subjects (severe versus mild COVID-19), GFAP, NfL and UCH-L1 had statistical significance difference (p ≤ 0.01), but not total TAU. Our findings corroborate previous studies that have also investigated NfL and GFAP levels in severe and mild COVID-19 patients in comparison to health control individuals. NfL levels were significantly associated to severity and neurological manifestations of COVID-19.[8, 22, 25] Kanberg et al (2020) investigated and followed up 47 COVID-19 subjects and health controls for GFAP and NfL levels and observed that, among severe patients, GFAP levels decreased along time but NfL remained constant after 11.4 days.[22] Unfortunately, we could not follow-up ICU subjects due to logistical resources, but we could also observe the relevant difference between ICU subjects versus mild COVID-19 and health control. Apparently, NfL levels tends to increase along time in severe/critical cases once it is related to neuronal injury and disease progression, while GFAP may indicate the astrocytic activation and injury as a first CNS response to infection.[22] The elevated results in GFAP described in our study are probably justified by sample collection in early stages of the disease. Similarly, Passos et al (2022) suggest that GFAP is upregulated by SARS-CoV-2, demonstrating astrocyte disintegration and promoting the overexpression of inflammatory mediators related to neurological injury and degeneration.[13]
In our study, we did not observe statistical significance in biomarkers levels when mild COVID-19 patients and health controls were compared. Different from our findings, a previous analysis of 405 mild COVID-19 adolescents and young adult subjects showed significant difference between NfL and GFAP levels when these were compared to health controls.[26] Probably, for mild COVID-19, the age decades influenced the results.
Regarding TAU and UCH-L1, we found significant higher levels of these two biomarkers among severe COVID-19 patients when compared to mild COVID-19 subjects and health controls. Interestingly, even analyzing other outcomes (neurological involvement), significant elevations in TAU, NfL and GFAP levels were also observed among hospitalized patients with and without neurological manifestations of SARS-CoV-2 infection.[27] These findings probably reinforce the early role of nervous system involvement as marker of disease severity,[28] as we previously demonstrated.[4, 10] TAU levels have also been associated to critically illness polyneuropathy/myopathy,[28] a relevant biomarker of severe axonal damage.[29] De Lorenzo et al (2021) investigated 104 subjects and reported a significant difference in NfL and UCH-L1 levels between ICU and non-ICU COVID-19 patients.[12] Conversely, they did not find the same significance for GFAP and TAU levels, presumably due to different population, which included not only mild but also moderate patients in the non-ICU group.
Focusing on the severe/critical COVID-19 group, from 108 included subjects, 37.96% (41) had a death outcome and 23 out of 41 were included in the biomarkers analyses. We found significant association among higher levels of NfL (p ≤ 0.001), GFAP (p < 0.05), TAU (p < 0.05) and UCH-L1 (p < 0.001) with death. This suggests the intense nervous system involvement, previously attributed by us and other authors, due to extended inflammatory response, axonal and neuronal injury, and astrocyte activation and damage.[30] GFAP and UCH-L1 are well-described biomarkers of traumatic brain injury, but when they are in higher levels concomitantly, they confer stronger evidence of severity in brain injury.[15]
It is important to reinforce that, in our study, all investigated biomarkers were already reported significantly higher early in ICU admission for subjects with a further lethal outcome, as described by other authors.[5, 12, 31–33] As well as the present study, other authors also found significant higher NfL levels in samples collected within the first five days in ICU, reinforcing this biomarker as early severity prognostic factor, and not a consequence of long-term hospitalization. [31, 33]
Concomitantly, GFAP and UCH-L1 levels were found significantly higher among deceased patients, when compared to severe survival COVID-19 patients. Regarding GFAP, increased levels have been previously associated to critical and lethal outcomes in severe COVID-19.[12, 13, 27, 34] Since astrocytes play an important role in brain response to viral infections, such as SARS-CoV-2, and inflammation, GFAP can be an important tool to predict astrocytic and CNS damage and neural injury [13, 34–37]
When it comes to UCH-L1, Tokic et al (2022) investigated severity biomarkers and testosterone in severe COVID-19 male patients, then suggested that UCH-L1 may be related to neurological deficits in such population, reinforcing that this may be a relevant prognostic biomarker of neurological manifestation of COVID-19.[38]
Finally, corroborating our findings, TAU protein has been previously described closely related to death in severe COVID-19.[12, 27] Apparently, SARS-CoV-2 infection may lead to hyperphosphorylation of TAU due to activation of both inflammatory signaling and oxidative stress pathways.[39]
It’s interesting that previous studies that investigated the same biomarkers presented slightly different results, reporting the significant difference in NfL, GFAP an TAU levels between survivors and deceased severe COVID-19 patients, but they did not observe the same to UCH-L1 levels.[12, 27]
We also investigated blood biomarkers from severe COVID-19 patients and we observed that Ferritin Peak was significantly higher (p < 0.0001) among deceased subjects in comparison to survivors. Ferritin levels were previously associated to neuropathic pain after severe or longer COVID-19 and to death.[8, 9] Frontera et al (2021), however, did not observe the same correlation amid ferritin and death and neither found significant association among ferritin and GFAP, NfL, TAU and UCH-L1.
It is also important to add that we only investigated peripheral blood in order to suggest an alternative non-invasive relevant biomarkers evaluation in a practical way using an ultrasensitive technique. The use of peripheral blood samples to investigate those biomarkers has been lately described by other authors,[40] which investigated biomarkers and proinflammatory cytokines in serum and CSF for comparison. These previous researches validated peripheral blood sample collection for this purpose an even observed an increase in NfL levels in serum that was not observed in CSF samples.
When we look for the virus role in disease severity, viral load could be an important indicative of severity or tissue damage by viral replication. In general, in naso-oropharingeal samples, SARS-CoV-2 viral loads tend to increase reaching a peak by the symptom onset, decreasing until the viral clearance in two weeks. Meanwhile, they use to persist in stool, but the clinical significance of this is not clear yet.[6, 7] However, in our findings, we could not find a significant association of SARS-CoV-2 viral loads to COVID-19 severity and lethality. Although some authors relate SARS-CoV-2 viral load to the extent of host inflammatory and immune responses and report the presence of SARS-CoV-2 in nervous system,[41] both peripheral and central,[28, 42] this direct relation is not clear. Jiao et al (2021) suggest that this virus crosses the blood-brain barrier, infecting neurons as observed in non-human primates.[43] However, other studies involving animal models could not detect viral particles in brain tissue of adult Syrian hamsters.[23] In human patients, Edén et al (2022) detected SARS-CoV-2 genomic RNA in plasma but not in CSF, N-antigen, an important replication biomarker, was also widely detected in plasma but not in CSF, for S-antigen it was rarely found in CSF.[44] Although in our study, patients were not examined for the assessment of viral load in the CSF, the levels of plasma biomarkers analyzed here are correlated and validated with the levels in the CSF. Therefore, these findings corroborate our results that higher viral loads seem not be related to severity outcome.
Thus, we can conclude that ferritin, NFL, GFAP, UCH-L1 and TAU are early biomarkers of the severity of SARS-COV-2 infection and may play an important role in therapeutic decision-making in the acute phase of the disease.