Critical illness polyneuropathy and myopathy in COVID-19 patients: a prospective observational intensive care unit cross-sectional cohort study

Background : Several reports on neurological complications associated with SARS-CoV-2 infection have been published. However, systematic description on intensive care unit acquired weakness (ICUAW) are still missing. Methods : The objective was to determine the incidence and characteristics of critical illness polyneuropathy (CIN) and myopathy (CIM) in patients with severe COVID-19. We also aimed to describe the electrophysiological features and their relation to plasma biomarkers for neuronal injury. This was a prospective observational intensive care unit cohort study. All adult patients admitted to the general intensive care unit (ICU) at Uppsala University Hospital, Uppsala, Sweden, between March 13 and June 8, 2020 were screened for inclusion. (GFAp) CIN/CIM those who did


BACKGROUND
Neurological manifestations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are increasingly recognized as a major complication with potential long-term consequences for both patients and the health care system [1]. The first study from Wuhan reported neurological symptoms in approximately 36% of confirmed coronavirus disease 2019 (COVID- 19) cases. However, the clinical investigation were mainly based on subjective symptoms and clinical/laboratory analyses [2]. Patients with severe COVID-19 infections are at risk to develop neurological manifestations, especially acute cerebrovascular disease, disturbed consciousness and skeletal muscle injury [2]. Manifestations in the peripheral nervous system have been less frequently reported but require specific investigations that may not always have been available or practical in the setting of the ongoing pandemic. In the previous severe acute respiratory syndrome (SARS) infection pandemic in 2004, a case series of four patients highlighted possibly coexistent critical-illness polyneuropathy and/or myopathy, suggesting further studies to determine a potential relationship between SARS coronavirus and neuromuscular problems [3]. Acute inflammatory polyneuropathy, e.g., Guillain-Barré syndrome (GBS), has been described in 19 COVID-19 patients worldwide [4][5][6], but to our knowledge intensive care unit acquired weakness (ICUAW) has not yet been characterized in COVID-19 patients.
Important pathophysiological mechanisms of ICUAW are critical illness neuropathy (CIN) and myopathy (CIM), where patients present with flaccid limb muscle weakness and failure to wean from the ventilator [7]. One of the medical challenges with COVID-19 patients is the high number of patients requiring prolonged mechanical ventilation in the intensive care unit (ICU) in combination with unusually high sedation requirements that both predispose to ICUAW [8]. CIN and CIM are important to identify, since survivors often present with severe residual disability and persistent exercise limitations several years afterwards [9][10][11].
There is further a clear distinction between outcome in CIN versus CIM, where patients with CIN have a slower or incomplete recovery, and higher mortality rate, whereas patients with CIM often show complete recovery within 6 months [12]. Thus, it would be highly valuable to predict COVID-19 patients at risk of developing CIN/CIM and secondly distinguish between the two if possible. So far, only one case report of a patient with CIM has been published [13] and no reports have to our knowledge described any cases of CIN associated with COVID-19. Except for electrophysiological parameters that classify CIN or CIM, increased plasma concentration of the axonal injury marker neurofilament light (NfL) has been described elevated in patients with ICUAW [14].
The aim of this study was to conduct a prospective study of all COVID-19 patients admitted to the ICU to characterize the ICUAW into CIN and CIM, as well as to determine potential correlating plasma biomarkers for neuronal injury. In order to understand the potential influence of COVID-19 on incidence of CIN/CIM, we also compared the number of cases with retrospective electrophysiological data for ICU patients during 2019.
positive SARS-CoV-2 [15]. ICU controls (non-COVID-19 patients) were treated for other disorders during the same time period and referred for electrophysiological evaluation due to ICUAW referring diagnosis to the department of clinical neurophysiology of either CIN (ICD-10 code G63.9) or CIM (ICD-10 code G72.9).
In order to compare the incidence rate of ICUAW and CIN/CIM during 2020 with a year prior to COVID-19, all cases admitted to the ICU were noted, in addition to diagnoses of those that were referred to electrophysiological evaluation.
The study was approved by the National Ethical Review Authority (EPM; No. 2020-01623).
Informed consent was obtained from the patient, or next of kin if the patient was unable give consent. The Declaration of Helsinki and its subsequent revisions were followed. The protocol of the study was registered (ClinicalTrials ID: NCT04316884). Electrophysiological data collected from COVID-19 patients and ICU controls were approved by the Ethical Review Board (case no 2015/105 and 2020-03377).

Electrophysiological examinations and parameters
Motor NCS (NCS) were performed in the median and ulnar nerve of one arm and in the fibular and tibial nerves of both legs; parameters including distal motor latency, conduction velocity, amplitude and amplitude decay between proximal and distal stimulation (conduction block assessment), as well as F responses. Orthodromic sensory NCS were done for the ulnar and radial nerves of one arm as well as the sural and superficial fibular nerves in both legs with parameters including distal latency, amplitude and conduction velocity. Combined motor and sensory amplitude scores were calculated as the mean amplitude of all recorded motor nerves divided by amount of nerves examined.
Low frequency (3 Hz) repetitive nerve stimulation was done of the ulnar nerve with recording over the adbuctor digiti quinti muscle, to eliminate decremental response as a lingering neuromuscular blocking effect of medications.
Electromyography with a concentric needle electrode was performed in limb and facial skeletal muscles in order to detect abnormal spontaneous activity at rest (fibrillations and/or positive sharp waves) and if possible, if the patients could collaborate, analyse the presence of myopathic (polyphasic, short duration, low amplitude) motor unit potentials.

Diagnostic criteria of CIN and CIM
Diagnostic criteria for both CIN and CIM included: i.
the patient was critically ill (multiorgan dysfunction and failures) and ii. limb weakness or difficulty weaning from ventilator after non-neuromuscular causes such as heart and lung disease have been excluded.
For CIN, additional diagnostic criteria included: iii. electrophysiological evidence of axonal motor and sensory polyneuropathy, iv. absence of a decremental response on repetitive nerve stimulation (RNS) [16].
For CIM, additional criteria included: iii. CMAP amplitudes 80% of the lower limit of normal in 2 nerves without conduction block; iv. sensory nerve action potential amplitudes 80% of the lower normal limit; v. Needle electromyography with short duration, low-amplitude motor unit potentials with early or normal full recruitment, with or without fibrillation potentials in conscious and collaborative patients; or increased CMAP duration or reduced muscle membrane excitability on direct muscle stimulation in noncollaborative patients; vi. absence of a decremental response on RNS [17].

Analysis of plasma biomarkers for neuronal injury and astrocytic activation
Plasma samples were collected at different time points in the ICU ward, early ( 9 days from admission) and at a later stage ( 11 days from admission). The results from COVID-19 patients with CIN/CIM (N=11) were compared with those from intubated COVID-19 patients treated > 12 days in the ICU who did not develop CIN/CIM (N=7). Levels of neurofilament light chain (NfL), tau and glial fibrillary acidic protein (GFAp) were measured using Single molecule array (Simoa) technology (Quanterix, Billerica, MA), as previously described [18] using a single batch of reagents. Intra-assay coefficients for internal control samples were below 7% for all analytes.

Statistical analysis
Data are represented as median (IQR) or n (%). Mann-Whitney U test was used for continuous parameters and the chi-square test for categorical parameters.
The plasma biomarker data were log transformed to achieve near normal distribution and analyzed with two-way repeated measures analysis of variance (ANOVA; CIN/CIM y/n x time) followed by Fishers LSD test or Bonferroni-corrected planned comparisons when suitable. Correlation analysis between plasma biomarkers (tau, GFAp, NfL) with NCS parameters were done with Spearman Rank correlation. Statistical comparison of descriptive variables (diagnoses) between the groups was done with Chi2-test. A p-value of < 0.05 was considered significant.

Characteristics of the ICU patients
After screening of 122 patients, a total of 111 COVID-19 patients were included in the study.
Demographic characteristics and comorbidities of these patients are displayed in table 1.
There was a significant overrepresentation of men in the CIN/CIM patient cohort as well as a higher BMI (table 1). There was no significant difference in age, blood group or preexisting comorbidities between the groups ( All patients but one in the COVID-19 cohort (13/14) were examined with a CT scan of the head at least once, and seven of these were also examined with an MRI of the brain. Four patients had pathological neuroradiological findings that did not explain the peripheral weakness: acute necrotizing encephalopathy (N=1), bilateral infarcts in deep white matter (N=1), unspecific white matter changes with microhemorrhage (N=1), and numerous bilateral microhemorrhages in white matter (N=1). The remaining patients had normal neuroradiological findings.

Comparison of incidence data on ICUAW 2019
Next, we aimed at comparing the incidence rate of ICUAW with a year prior to COVID-19.

Analysis of blood biomarkers for neuronal injury and astrocytic activation
Analysis of biomarkers for neuronal injury and astrocytic activation was done from early plasma samples, within 9 days (median: 4, range: 3-9), and late, after 11 days (median: 16,  Figure 1C).

Correlation between plasma biomarkers and NCS parameters comparing a subgroup of non-CIN/CIM with CIN/CIM
In  Figure 3B).

DISCUSSION
There was a strikingly higher incidence of CIN in the COVID-19 ICU cohort compared with the non-COVID-19 ICU cohort during the enrolment period. This is in contrast to the overall literature, which states that CIM is more common than CIN [19]. One recent case of CIM as a likely consequence of SARS-CoV2 infection was published [13], but to our knowledge no cases of CIN have yet been reported. Speculations have also been made regarding increased number of CIM patients in COVID-19 cohorts since propofol is considered a risk factor for CIM [20]. Since patients with CIN has a slower recovery or do not recover in addition to higher mortality rates, this is an important ICU consequence to consider for the COVID-19 patients. Depending on the underlying comorbidities, CIN and CIM can be common in critically ill patients. In patients suffering from sepsis with multiorgan failure the incidence can be as high as 100% [21]. In patients with ARDS, the incidence is about 20-60% [22,23] and in general about 20% [24]. There are no available blood biomarkers to distinguish CIN or CIM from normal NCS in patients with ICUAW. A previous pilot study observed that peak neurofilament levels had good discriminative power for ICUAW but this occurred at a later stage and first after the muscle strength assessment [14]. In a recent study higher plasma levels of NfL and GFAp were correlated with severity of COVID-19 [18]. All our included subjects suffered from severe COVID-19, but still the CIN/CIM patients had higher plasma NfL levels. In the current study, we observed significantly higher NfL levels in the CIN/CIM group, both at the early and late time points, prior to onset of ICUAW. NfL is a neuronal cytoplasmic protein, which is particularly highly expressed in myelinated axons. Levels of NfL in the blood proportionally increase to the degree of axonal damage in several neurological disorders, including multiple sclerosis, amyotrophic lateral sclerosis and Alzheimer's disease [29], but also in peripheral neuropathies [30][31][32]. Several patients in our control group had underlying cerebral insults that could affect the biomarker levels but still the CIN/CIM showed significantly higher levels of NfL. A larger study would be needed to confirm the potential of NfL to determine peripheral axonal loss in CIN or other polyneuropathies. GFAp is a suggested astrocyte biomarker for glial activation, with elevated levels in Alzheimer's disease [33]. Early high levels of GFAp were also found in the CIN/CIM group, which also correlated with motor amplitude score, indicating that GFAP could have potential as marker for axonal pathology in myelinated nerves. Tau levels were slightly higher among CIN/CIM patients, which is interesting since tau mainly is a biomarker in cerebrospinal fluid in neurodegenerative brain disorders [34]. We found a correlation between tau and the sensory amplitude score, which is in line with a recent study indicating that serum levels of phosphorylated tau may be a useful biomarker for severe axonal injury due to traumatic spinal cord injury [35].

Strengths and limitations
This observational study has several strengths. First, we prospectively included all patients to the ICU in an unbiased manner and recorded all vital parameters for the duration of the ICU care. All patients referred for electrophysiological evaluation were examined according to the same standardized protocol with NCS and EMG.
This observational study also has several limitations that may affect the generalizability of our results. First, referrals to NCS and EMG were strictly selected to patients with quadriplegia, which could have resulted in a false low incidence of CIN/CIM. Also, approximately 1/5 of patients died within 2 weeks from ICU admission and this probably affected the incidence rate of CIN/CIM. Second, there is a higher proportion of men than women in the COVID-19 cohort, however this predominance of men who acquire a more severe form of the disease is known [36]. Third, the study was done in only one Swedish hospital. Since health-care systems along with treatment strategies vary between hospitals and countries, it is not certain that results from high-income countries are directly transferable to low-income and middle-income countries. The finding of elevated NfL and GFAP in patients who later developed CIN/CIM needs to be validated in larger prospective studies, to know how these biomarkers would potentially influence the clinical decisions for these patients. Furthermore, a muscle biopsy was not done in the ICU, and thus the myosin loss as part of CIM could not be assessed.

CONCLUSIONS
In summary, we found a high proportion of COVID-19 patients with ICUAW who develop CIN. Unlike the previously described cases of COVID-19 patients developing GBS, where the infection most often precede GBS, it is most likely that the lengthy ICU care is an important explanation to the high incidence of CIN. This is important to consider in the differential diagnostic workup as well as the further rehabilitation of these patients.

Ethics approval and consent to participate
The study was approved by the National Ethical Review Authority (EPM; No. 2020-01623).
Informed consent was obtained from the patient, or next of kin if the patient was unable give consent. The Declaration of Helsinki and its subsequent revisions were followed.
Electrophysiological data collected from COVID-19 patients and ICU controls were approved by the Ethical Review Board (case no 2015/105 and 2020-03377).

Consent for publication
Not applicable.

Availability of supporting data
The datasets analyzed during the current study are available from the corresponding author on reasonable request.

Funding
The study was funded by the