Increased prevalence of mild myopathic changes in the post-COVID-19 duration

ABSTRACT Objective There are reports of peripheral nerve and muscle involvement during or after coronavirus disease 2019 (COVID-19), even following a mild infection. Here, we aimed to analyze the objective findings regarding peripheral nerve, neuromuscular junction, and muscle function using electrophysiology in patients with a previous COVID-19 infection. Methods All consecutive patients with a history of COVID-19 were questioned for post-COVID-19 duration-related neurological complaints via Composite Autonomic Symptom Score-31 (COMPASS-31), modified Toronto Neuropathy score (mTORONTO), and Fatigue Severity Scale (FSS). Patients were dichotomized into two groups based on their scores in the questionnaire. Group 1 (patients with high scores in any area of the questionnaire) and Group 2 (patients with normal scores in all sections of the questionnaire). In the second step, Group 1 was invited to a preplanned hospital visit for electrophysiological analysis, including nerve conduction studies, repetitive nerve stimulation, needle electromyography (EMG), quantitative motor unit potential analysis (qMUP), and single fiber EMG. We included 106 patients in the study. According to the questionnaire, 38 patients constituted Group 1, and 68 formed Group 2. Results Of the 38 patients, 14 accepted and underwent preplanned electrophysiological examinations. Needle EMG revealed small, short, polyphasic MUPs with early recruitment, and qMUP analysis demonstrated an increased percentage of polyphasic potentials in three patients. The examinations in other patients were unremarkable. Conclusions The high prevalence of complaints and objective myopathic findings in our cohort implicated the role of muscle involvement in the post-COVID-19 duration. Considering the socioeconomic and psychological burden of the post-COVID-19 duration among individuals and societies, a better understanding of the symptoms and myopathy is warranted.


Introduction
Since 2019, the acute coronavirus disease 2019 (COVID- 19) resulted in more than 639 million cumulative cases and 6 million fatalities [1].Post-COVID -19 syndrome refers to the signs and symptoms that continue or develop after acute COVID-19, covering the period after the 12 th week [2].
Among the constellation of the sign and symptoms, neurological manifestations include headache, cognitive and behavioral disturbances, sleep disorders, fatigue, myalgia, and autonomic findings [3,4].Fatigue and myalgia represent the most common and impactful extra-pulmonary symptoms, as fatigue is reported in 35-77% and myalgia in 6-16% of cases [4][5][6].
Diagnosis of the post-COVID-19 syndrome solely depends on symptoms and ruling out alternative diseases due to the absence of objective diagnostic tools [7,8].According to reports and patient experiences, this causes some professionals to approach the entity in a highly septic manner, stigmatizing post-COVID-19 syndrome patients as hypochondriacs and ascribing the syndrome to a psychological state [9].
Contrarily, there are reports objectively demonstrating myopathic involvement in patients during the acute and post-acute COVID-19 disease.In a study conducted in hospitalized patients without muscular complaints, %50 had myopathic changes in needle electromyography (EMG) [10].In another study, peripheral nerve and muscle involvement was demonstrated using electrophysiology in patients with a history of COVID-19, even when asymptomatic [11].Although there are reports of critical illness myopathy following severe COVID-19, opposing cases with extreme fatigue and weakness, with myopathy in muscle biopsy, following a mild infection, are also present [12,13].These reports suggest that muscle involvement might be a component of the disease, apart from the severity.However, iCOMPASS-31, >four in mTORONTO, and > 2.3 in these investigations, patients were examined electrophysiologically during either the acute phase or the first weeks following acute COVID-19 infection; consequently, the post-COVID-19 syndrome period was not covered [2].
Here, we aimed to investigate the neurological symptoms in the post-COVID-19 duration and analyze the objective findings of peripheral nerve, neuromuscular junction, and muscle function using electrophysiology in our cohort.

Patients and methods
In the first step, all consecutive patients admitted to COVID-19 outpatient clinics who had a confirmed negative PCR test after the acute infection period were invited to the study.The invitation included an online questionnaire sent to all eligible subjects between October-November 2021.
The local ethical committee approved the study (approval number: 405283).All participants gave informed consent.

Study Groups
Results of the questionnaire were checked, and two subgroups were constituted: patients with high scores in any of the scorings mentioned earlier (Group 1) and patients with normal scores in all sections (Group 2).
In the second step, Group 1 was invited to a preplanned hospital visit for neurological and electrophysiological evaluation.Here, a detailed history and examination were performed by a neurology consultant.Afterward, the electrophysiological assessment was performed by two experts with more than ten years of experience, who were both blind to the patients' complaints until the end of the session.For this purpose, a predesigned electrophysiological evaluation was conducted for all participants, though it was expanded depending on electrophysiologists' judgment regarding pathology.

Electrophysiological Evaluation
The electrophysiological evaluations were performed using the Keypoint EMG device (Dantec, Denmark).They included nerve conduction studies (NCS), repetitive nerve stimulation (RNS), single fiber electromyography, needle electromyography, and quantitative motor unit action potential analysis (qMUP analysis).
Electrophysiological findings were compared with a control group of healthy subjects of similar age and sex.
• Nerve conduction studies (NCS): We performed motor NCS of bilateral median and ulnar nerves and bilateral peroneal and tibial nerves using bar electrodes.The recording electrode for median motor NCS was on the abductor pollicis brevis muscle; the stimulation was done at the wrist and antecubital fossa.The recording electrode for ulnar motor NCS was on the abductor digiti minimi muscle; the stimulation was performed at the wrist, cubital tunnel, and 6 cm proximal to the cubital tunnel.The recording electrode for peroneal motor NCS was on the extensor digitorum brevis muscle, and the stimulation was at the ankle, 3 cm distal to the fibular head, and 5 cm proximal to the fibular head.The recording electrode for tibial motor NCS was on the flexor hallucis brevis muscle, and we stimulated behind the medial malleolus and popliteal fossa.The sensory NCS was performed bilaterally on the median and the ulnar nerves antidromically and unilaterally on the sural and superficial peroneal nerves.All recordings were done using supramaximal stimulations.All NCSs were performed following the methods for safety precautions, measurements, and electrode placement [17].Filter settings for sensory NCS were 20 Hz-2 kHz and for motor NCS 2 Hz-10 kHz.For sensory NCS, the sweep speed was 2 msec/div, and the sensitivity was 10 µV/div.For motor NCS, the sweep speed was 5 msec/div, and the sensitivity was 5 mV/div.
The evaluated parameters for motor NCS were distal motor latency, conduction velocity (CV), compound muscle action potential (CMAP) amplitude, and F-wave latencies.For sensory NCS, distal latency and sensory nerve action potential (SNAP) amplitude were evaluated.
• Repetitive nerve stimulation (RNS): Unilateral compound muscle action potentials (CMAPs) of abductor digiti minimi and trapezius muscles were recorded using surface silver -silver chloride electrodes.The recordings of CMAPs of the muscles mentioned above were made following previously published reports [18].A single square wave electrical stimulus of 0.2 milliseconds duration at the supramaximal intensity was applied to the ulnar and accessory nerves, respectively.RNS was applied using supramaximal ten stimuli at 2 and 3 Hz to the ulnar and accessory nerves at rest and supramaximal 20 stimuli at 15 Hz to the ulnar nerve following 10 seconds of exercise.
The signals were filtered between 3 kHz highcut and 2 Hz low-cut.
The evaluated parameters for RNS were the percent change of the area and amplitude of the 4 th CMAP at a frequency of 2 and 3 Hz at the rest and the area and amplitude of the 20 th CMAP at 15 Hz following 10 seconds of exercise.
• Needle electromyography (EMG): Needle EMG was performed with a 50-mm concentric needle electrode.The standard limb EMG included the assessment of biceps brachii (BB), extensor digitorum (ED), and tibialis anterior unilaterally.Analysis of insertional activity, spontaneous activity, and MUPs was performed according to the previous reports [19].The filter settings were 20 Hz high-pass, 10 kHz low-pass, 100 μV/division gain, and 10 ms/division sweep speed.
Firstly, insertional activity, fibrillation potentials, positive sharp waves, and fasciculation potentials during rest are assessed.Afterward, evaluation of MUP activity regarding morphology (duration, polyphasic, and amplitude), stability, and firing pattern (activation, recruitment, interference pattern) was evaluated.
The electromyographers noted their first decision if any interpretation of acute or chronic neuropathic or myopathic changes were detected before performing the quantitative MUP analysis.
• Quantitative MUP analysis (qMUP): qMUP analysis was performed from ED and BB muscles, and sampling of at least 20 MUPs during weak contraction was performed [20].Mean duration, amplitude, and percentage of polyphasic potentials were evaluated, and results were compared to healthy controls.• Single fiber electromyography (SFEMG): Rightsided ED and BB muscles were used for SFEMG jitter measurements with a 50-mm concentric needle electrode.Participants were asked to activate each muscle with slight voluntary contraction, and at least 20 apparent single-fiber action potentials were collected for each muscle and analyzed [21].The filter settings were 1 kHz highpass and 10 kHz low-pass.The jitter of ED and BB muscles was evaluated.
Muscle magnetic resonance imaging (MRI) and serum biochemistry were performed for those with abnormal findings in electrophysiological analysis.

Data and statistical analysis
Data analyses were performed using the SPSS 22.0 software statistical package.The distribution of the data was not normal.Demographic and clinical data of Group 1 and Group 2 were compared.The Chisquare test was used for qualitative data.Electrophysiological findings between patients and healthy subjects were also compared.The comparisons between patients and healthy subjects were made by the Mann-Whitney U test.A p-value ≤0.05 was considered significant.

Results
Demographic and clinical findings in the general study population: The questionnaire was sent to 172 patients, and 106 of them replied.These 106 patients created the study population.The mean duration from the negative PCR test to the questionnaire response time was 147 days (median: 104 days, min-max: 31-390 days).
Eighteen of 106 patients (%17) had chronic diseases.They regularly used drugs: hypothyroidism and thyroid replacement therapy (n = 4), diabetes mellitus with oral antidiabetic medications (n = 4), essential hypertension and use of amlodipine (n = 2), warfarin anticoagulation for previous valve replacement (n = 1), psoriasis with no regularly-used drugs (n = 1), asthma with inhaler steroids (n = 1), use of trazodone and paroxetine for insomnia and anxiety (n = 2), history of renal transplantation and use of ramipril, tacrolimus, prednisolone and mycophenolic acid (n = 1), lymphoproliferative diseases without treatment (n = 2).There were 38%35.8)patients with high scores in at least one of the scoring systems.They constituted Group 1, and the remaining 68 patients formed Group 2. Distribution of age, gender, and duration between negative PCR test and the time of response to the questionnaire were similar between Groups 1 and 2. Headache and myalgia were significantly more frequent in Group 1 compared to Group 2 (p = 0.001 and 0.021, respectively).Demographical data, accompanying neurological complaints, along with the scores in the questionnaire are shown in Table 1.
Electrophysiological findings of Group 1: Patients in Group 1 were invited for further evaluation.Among 38 patients in Group 1, 14 were evaluated, while the remaining 24 did not participate in the next step (in-hospital evaluation).Of 24, 18 patients were unwilling to participate due to improvement of their symptoms, along with the anxiety of reinfection by COVID-19 in the hospital environment, whereas 6 of 24 were excluded regarding influenza infection following COVID-19 in three, lymphoproliferative disease in two and pregnancy in one.
Among patients who underwent detailed neurological examination, all except one experienced COVID-19 infection at home.The only hospitalized patient did not require invasive ventilation or intensive care unit but needed O 2 support constantly during hospitalization.Five patients had chronic illnesses, psoriasis, hypothyroidism, renal transplantation, valvular replacement, and asthma.Three (%21) of 14 patients complained of headaches; they all had prior headache history.Thirteen (%92) patients described new-onset sensory complaints which occurred in the post-COVID-19 duration.Twelve (%85) patients complained about fatigue; two had severe fatigue, according to FSS, and five had myalgia.All had normoactive deep tendon reflexes and normal sensory examination.One patient had proximal muscle weakness, and the muscle strengths of remaining were normal.
NCSs, RNSs, and jitter analysis by SFEMG were normal in all 14 patients.Needle EMG revealed small, short, polyphasic MUAPs with early recruitment in three patients but normal remaining.qMUP analysis also objectively revealed an increased percentage of polyphasic potentials.When qMUP analysis results were compared with age and gender-matched healthy controls, mean amplitude and durations were similar between groups.In contrast, polyphasic potentials were substantially elevated in patients with post-COVID-19 syndrome (Table 2).
One of these three patients had proximal muscle weakness.All three had fatigue, and none of them had myalgia.There were not using any medications regularly that could cause the myopathic changes.Muscle MRIs with gadolinium of proximal upper and lower extremities in these three patients were normal (Figures 1 and 2).Serum biochemistry analysis, including creatinine kinase (CK), lactate dehydrogenase (LDH), erythrocyte sedimentation rate (ESR), and C reactive protein (CRP) were performed, and results were normal (CK: 84 U/L, 54 U/L, 61 U/L; LDH: 204 U/L, 134 U/L, 123 U/L; ESR: 3 mm/h, 9 mm/h, 11 mm/h; CRP 7.7 mg/l, 1.7 mg/l, 0.60 mg/l, respectively)

Discussion
Subtle myopathic changes in COVID-19 were first implied by Villa et.Al [10].They performed EMG within the 5 th -30 th days of the acute disease and found myopathic changes in half of the asymptomatic patients.They concluded that even in asymptomatic patients, neuromuscular involvement should be considered a part of the disease spectrum [10].Following that, a study by Tankisi et al. was the first to demonstrate myopathic changes in post-COVID-19 syndrome.More than half of the patients with neuromuscular complaints had objective myopathic changes in qMUP analysis, and they attributed these findings to fatigue and myalgia [22].After these preliminary results, they performed an electrophysiological evaluation on 85 patients with post-COVID-19 syndrome, and myopathic changes were consistently present in patients with neuromuscular symptoms [23].In line with these findings, patients with mild myopathic changes were present in our cohort following a mild acute infection course.
Although there are reports of diffuse muscle atrophy on muscle MRI of patients with critical illness myopathy following COVID-19, no reports have qMUP analysis was performed from the extensor digitorum and biceps brachii muscles for patients and controls.At least 20 MUPs during weak contraction were sampled for each muscle, and mean amplitude, duration, and percentage of polyphasic potentials were compared between patients and healthy controls.Polyphasic potentials were significantly higher in patients.(µV, microvolt ms, millisecond).*A p-value ≤0.05 was considered significant.evaluated patients with post-COVID-19 syndrome with myopathy after a mild or asymptomatic infection [24].For this purpose, patients in the present study were assessed further with blood biochemistry and muscle MRI.However, there was not any pathology.Muscular involvement of post-COVID-19 syndrome in our cohort could be related to dysfunction of physiological processes rather than structural abnormalities.In line, patients with post-COVID-19 syndrome following mild COVID-19 infection were evaluated by muscle ultrasound, which revealed no structural abnormalities despite clinically relevant muscle weakness following COVID-19 [25].However, this interpretation needs confirmation with further studies.
There are several studies evaluating muscle biopsy specimens of COVID-19 patients.In a study conducted by muscle samples of autopsies of patients who died after COVID-19, they concluded that inflammatory/immune-mediated muscle damage was related to the release of cytokines, and there was no evidence of direct SARS-CoV-2 invasion of muscle tissues [26].The study by Hejbol et al. aimed to reveal histopathological changes in post-COVID-19 syndrome patients with mild acute infection [27].They performed muscle biopsy in 16 patients with muscle weakness and myopathy in EMG, and all biopsy specimens had histopathological findings.They found a wide range of structural myopathic findings, including fiber damage, mitochondrial changes, inflammation, and capillary injury, and concluded that all of them could cause fatigue.On the other hand, these findings might be questionable for some experts, although implicating neuropsychological and emotional factors for casualty does not seem rational [28].Structural myopathic findings in these studies might seem contradictory as MRI findings were normal in our patients despite not including muscle biopsy.However, considering that the studies above included biopsy specimens either from autopsy samples or patients with muscle weakness and myopathy, our study population was composed of patients with relatively mild neurological complaints, which did not necessitate seeking medical care [26,27].Furthermore, there have been previous concerns about post-COVID -19 myopathy, which could be linked to either rhabdomyolysis or the non-use of muscles caused by physical inactivity [29].However, three patients with myopathic changes in EMG in our cohort were active in their daily life and working during the evaluation period.They also experienced acute COVID-19 infection as mild and eventless.Therefore, our findings could be interpreted as myopathic changes were observed in post-COVID-19 syndrome patients with mild complaints in proximal and distal extremity muscles via electrophysiology, even though imaging and laboratory investigations failed to provide additional information.
Another aspect of our findings that deserves attention was the lack of nerve conduction disorders despite the high prevalence of sensory complaints.This could be explained by minor nerve fibers dysfunction caused by COVID-19 infection rather than large fibers, and electrophysiological investigations in the present study did not evaluate small fibers [30].In line with this, our previous study demonstrated abnormal modulation of nociceptive pathways in post-COVID-19 syndrome patients leading to small fiber dysfunction [31].Moreover, although neuromuscular dysfunction was demonstrated in critically ill patients priorly, RNS results were normal in our study population, suggesting post-COVID-19 duration-related complaints are unrelated to neuromuscular junction [32].
Considering the discrepancy between the patient perception of complaints about post-COVID-19 duration and limited sources of objective diagnostic tools, diagnosis of post-COVID-19 syndrome is still subjective [33].However, our study suggests that electrodiagnostic evaluation of these patients, including qMUP analysis, could contribute to the diagnosis.Consequently, patients having post-COVID-19 syndrome with neurological complaints should undergo an electrodiagnostic assessment.

Limitations
Our study has limitations.First, our cohort was small, and these findings need confirmation with larger study groups.Second, the mean time between acute infection and evaluation was relatively short in our cohort differing between one month to one year.Evaluating patients with a wide range of duration from the acute infection and longitudinal follow-up could provide more information regarding the natural course.Another limitation might be that fatigue may be related to the central nervous system or any other end-organ involvement.Although this study does not contain an evaluation regarding these organs, patients were questioned regarding central nervous system complaints, and myopathy was present nevertheless.

Conclusion
This study scans patients with a recent COVID-19 infection.Therefore, it increases sensitivity by capturing relatively mild complaints that one may have but not seek medical care.Keeping this manner in mind, one in three patients having post-COVID-19 syndrome-related neurological complaints put forth the real burden of the patient experiences.More importantly, our data support the hypothesis that these symptoms might be associated with muscle, based on myopathic changes in needle EMG and qMUP analysis.Myopathic changes were observed in both proximal and distal extremity muscles, and imaging and laboratory investigations failed to provide additional information.Therefore, our results suggest that post-COVID-19 syndrome-related dysfunctional outcomes might be related to muscular involvement.
Considering the socioeconomic and psychological burden of post-COVID-19 syndrome among individuals and societies, a better understanding of the symptoms and related pathology is warranted.The high prevalence of complaints and objective myopathic findings in our cohort implicate a role of muscular involvement in the post-COVID-19 duration.

Figure 1 .
Figure 1.MRI images from the proximal mid crural level of the lower extremity demonstrate normal imaging features of the muscles.a) Fat -suppressed T1 weighted axial image with contrast, b) Fat-suppressed T1 axial image, c) T1-weighted axial image.

Figure 2 .
Figure 2. MRI images of the forearm demonstrate normal imaging features of the musculature.a) T1-weighted axial, b) Fatsuppressed T2 axial image.

Table 1 .
Demographical and clinical data of the study population.
*A p-value ≤0.05 was considered significant.

Table 2 .
Comparison of qMUP parameters between patients and controls.