Effect on Quality of Life of Therapeutic Plasmapheresis in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome Patients with Elevated Ꞵ-Adrenergic and M3-Muscarinic Receptor Antibodies – a Pilot Study

1. Introduction

The COVID-19 pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to a global health crisis with profound immediate and long-term consequences. While the acute phase of COVID-19 has been well-documented, increasing attention is being directed toward the persistent, debilitating symptoms experienced by a subset of patients well beyond the initial infection period. These prolonged symptoms fall under the umbrella term Post-COVID-19 Syndrome (PCS), also referred to as Long COVID [1]. PCS encompasses a wide range of symptoms, including fatigue, exertion intolerance, cognitive impairment, orthostatic intolerance, and autonomic dysfunction, which can persist for months or even years after the acute infection has resolved [1].

Epidemiological studies suggest that approximately 5-10% of individuals who recover from the acute phase of COVID-19 develop PCS. The clinical presentation of PCS is highly variable, with symptoms overlapping significantly with those of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS)[1,2]. This overlap includes characterized symptoms such as profound fatigue, post-exertional malaise (PEM), myalgias and arthralgias, sleep disturbances, neurocognitive impairment, and autonomic dysregulation [1,2]. The pathophysiological mechanisms underlying PCS are complex and multifactorial, involving immune dysregulation, autoimmunity, endothelial dysfunction, viral persistence, and reactivation of latent viruses such as Epstein-Barr virus (EBV) [2].

Recent research has highlighted the role of immune activation and dysregulation in PCS, with several studies reporting the presence of autoantibodies, including those targeting G protein-coupled receptors (GPCRs) like the ß2-adrenergic receptor [1]. These autoantibodies have been associated with symptom severity and functional impairment in both PCS and ME/CFS [3]. Additionally, endothelial dysfunction, characterized by inflammation, microclots, and hypoperfusion has been identified as a significant contributor to the persistent symptoms observed in PCS [4].

Fatigue is a main symptom of PCS, it is in the literature well documented, that there are significant disturbances affecting numerous functions such as sleep, cognitive dysfunction, anxiety, depression, impairment of executive functions, psychomotor coordination. Patients with long COVID may suffer from brain hypometabolism, aligned with hypoperfusion of the cerebral cortex and changes in the brain structure and functional connectivity [5].

ME/CFS is not just significantly overlapping with PCS, recent studies implicate that the chronification of PCS could possibly fulfill the criteria of ME/CFS (one of the main diagnostics criteria of ME/CFS is an at least 6 months persistence of the symptoms) [6,7]. Therefore, further research is needed to find proof that there is not only a significant clinical and pathophysiological overlap, but shared therapeutic possibilities of ME/CFS and PCS could also exist. Despite growing knowledge about the mechanisms of PCS, unfortunately, there is limited evidence based data on effective curative therapies available today. In this observational study, we aimed to present how the wellbeing develops of our patients during and after the extracorporeal PE + IVIG treatments.

There are assumed to be three key steps underlying the initiation and persistence of ME/CFS [12]:

• Triggering Immune Response—An infection-induced immune response acts as the initial trigger, with B cells, plasma cells, and autoantibodies playing a role in the underlying pathology.
• Vascular and Autoantibody-Mediated Dysfunction—Autoantibodies, particularly those targeting GPCRs, may affect the vascular system, leading to endothelial dysfunction, impaired neurovascular control, and autonomic small nerve fiber involvement. These autoantibodies—whether pathogenic IgGs or functional autoantibodies that persist beyond the usual resolution—disrupt homeostasis. This disturbance manifests as endothelial dysfunction in both large and small arteries, impaired venous return, preload failure, and arteriovenous shunting, ultimately contributing to blood flow dysregulation and exertion-induced tissue hypoxia.
• Secondary Compensatory Mechanisms—In response, the body adopts compensatory adaptations, including increased sympathetic tone and metabolic shifts aimed at maintaining energy supply. These adaptations further shape the clinical presentation and symptomatology of ME/CFS.

There are numerous potential therapeutic strategies targeting these pathways [3]:

• Interfere with the pathological immune response:
  • with B-cell depletion therapy (anti-CD20 antibody)
  • use of cytotoxic drugs (cyclophosphamide)
  • modulate the plasma cell survival factors (Anti BAFF antibody)
  • Plasma cell inhibition (Anti-CD38 antibody, proteasome inhibition)
  • or through Immunoglobulin manipulation (neonatal fragment crystallizable receptor (FcRn) targeting, immunoadsorption, IVIG)
• Address the vascular dysregulation: endothelial dysfunction, arteriovenous shunting, impaired autoregulation (plasmapheresis had been proven to improve endothelial dysfunction in critically ill patients with Disseminated intravascular coagulation (DIC) [12])
• Support the patient’s compensatory adaptation by pacing therapy [13], acetylcholinesterase inhibition or cognitive techniques.

Our study group decided to address all three major potential therapeutic strategies of the ME/CFS patients, firstly by applying plasmapheresis for improving the endothelial dysfunction and platelet function, secondly in term of administering IVIG to regulate the derailed, inadequate immune response, and thirdly by performing pacing therapy to support the compensatory adaptation mechanisms.

Even if there is no reliable research data at the current date that PE could improve the clinical outcome by extracting the microclots [14] that have been identified in PCS and ME/CFS patients, it has been proven that PE can reduce IgG levels [15] and can improve endothelial dysfunction and platelet function [12]. Given the better availability and lower cost of PE than immunoadsorption, positive results with PE-technique could provide a treatment option even for lower-income countries in demanding need for ME/CFS therapy.

2. Materials and Methods

2.1. Study Protocol

Adult patients (≥18 years) with elevated autoantibodies (ꞵ1, ꞵ2, M3, M4), measured between January 2023 and September 2024, were enrolled in this pilot study at a Swiss tertiary care hospital. The patients had typical chronic fatigue symptoms like exertion intolerance, post-exertional malaise, orthostatic dysregulation, concentrating difficulty, brain fog, headache, insomnia, high resting pulse, some even elevated body temperature or pain. We worked together with the Outpatient Clinic of Internal Medicine (Long COVID consultation) and the Clinic of Nephrology and Transplant Medicine (extracorporeal therapies, i.e., PE).

The patients went through a comprehensive work-up before being referred to plasmapheresis. Other possible causes that can mimic ME/CFS-symptoms have been ruled out: cardiac testing by echocardiography, in some cases also bicycle ergometry or cardiac MRI. Testing for early-onset neurodegenerative diseases by functional testing with the Montreal Cognitive Assessment (MoCA) and/or neuroimaging cerebral computed tomography (CT) or cerebral magnetic resonance imaging (cMRI) scans to rule out demyelinating diseases. Functional pulmonary testing by body plethysmography and carbon monoxide (CO) diffusing capacity testing have been performed, in a few cases also chest imaging to assess possible pulmonary embolism, and sleep apnea screening for rule-out this possibility as well. Every patient completed a 6-minute walk test (6MWT) and a Schellong test as a baseline assessment. Some patients had been examined for possible gastrointestinal, rheumatological, and for other neurologic causes of their symptoms.

Prior to the referral for PE, the patients were individually treated with various conservative therapeutic measures (professionally instructed pacing therapy, physiotherapy, psychotherapy, tricyclic antidepressive, Serotonin–norepinephrine reuptake inhibitors (SNRIs), selective serotonin reuptake inhibitors (SSRIs), H1-antihistaminergic agents, ritaline, antiepileptics, naloxone, and in some cases even inpatient rehabilitation).

Inclusion criteria were clinically diagnosed CFS/ME with elevated GPCRs. Other pilot studies have shown an effect of repeated immunoadsorption on CFS/ME patients [2], therefore we decided to perform PE with intravenous immunoglobulin (IVIG)- infusions as in the Kiprov-case [16] as an off-label therapy. This method could also remove the autoantibodies and may remove the microclots as well. Newest research implicates that in ME/CFS cases a dysregulation of the coagulation system is present, the endothelial dysfunction, and the downregulation of complement machinery can be assessed on a molecular level [17].

However, there are some raising concerns about the pathophysiological role of microclots in developing ME/CFS, implying that microclots do not play a significant role in the pathomechanism [14].

For PE, we used the machine Spectra Terumo Optia Apheresis System. For the exchange we gave albumin 5%, one time of the plasma volume. A total of 4 treatments (on day 1 and 5, and in 1 to 4 months interval) were performed. We measured the autoantibodies before and two weeks after the fourth (in two cases after the third) plasma exchange. At the Outpatient Clinic of Internal Medicine the patients received standardized tests for the assessment of physical function and symptoms in a standardized manner (interdisciplinary workflow, Table 1).

At the end of every PE session the patients received additionally 2 Grams of Octagam^Ⓡ intravenous independently of their body weight. Human immunoglobulins are meant to have an immunomodulatory and immunosuppressive effect, even in low dosage [18].

All of the patients were regularly monitored at the Long COVID Consultation during and after the series of 4 plasma exchange therapies. All of the collected data was documented in the patient management system (PMS) of our hospital.

The ISI (Insomnia Severity Index), FSS (Fatigue Severity Scale), HADS (Hospital Anxiety and Depression Scale) and EQ-5D-5L (European Quality of Life 5 Dimensions 5 Level Version) questionnaires had been applied to assess the symptoms of the patients with standardized methods. The EQ-5D-5L questionnaire has been developed by the EuroQol Group and it is a well studied tool for assessing the Quality of Life. The EQ-5D-5L descriptive system comprises five dimensions (mobility, self-care, usual activities, pain / discomfort and anxiety / depression). Each dimension has five response levels: no problems, slight problems, moderate problems, severe problems, unable to/extreme problems. The index-value is calculated by deducting the appropriate weights from 1, the value for full health [19].

2.2. Study Procedure and Data Collection

All four autoantibodies were measured two times per patient over the course of the study at the measurement time points: baseline before starting PE, and 2 weeks after the last PE. Clinical outcomes–consisting of the 6MWT, Schellong test, as well as psychometric assessments with the ISI (insomnia), FSS (fatigue), HADS (depression and anxiety) and EQ-5D-5L (quality of life) questionnaires–were measured three times at T1, T2, and T3. See Appendix A Table A1 for psychometric properties of the validated questionnaires used in this study.

2.3. Statistical Analyses

Concerning a potential main trial, the study design requires a total of 56 patients for an expected 80% statistical power based on an a-priori power analysis calculated with the SIMR package v.1.0.7 using Monte Carlo simulations [27]. The power analysis is based on a multilevel model (MLM) with two measurement time points for each patient and each of the two investigated variables, namely antibody concentration and clinical outcome measures. We expected a medium to large effect size R2 of 0.16 as comparable studies investigating the effect of plasma exchange treatments in ME/CFS patients with elevated β2-adrenergic receptor autoantibodies [28], and adrenergic dysfunction in ME/CFD patients [29] reported medium to large effect sizes. Cocks and Torgerson (2013) recommend having at least 9% of the sample size of the main planned trial when conducting a pilot study, resulting in 5 patients for the present pilot study [30].

Statistical analyses were performed using R Project for Statistical Computing v4.4.2 [31]. After inspecting incomplete data by using the VIM package v6.2.2 [32], we imputed missing values with the MICE package v3.17.0 based on the multivariate imputation by chained equations algorithm for the missing at random (MAR) scenario [33]. After imputation, we evaluated the imputed data using convergence diagnostic tools such as trace and density plots. Estimates of model parameters and weights obtained in the imputed datasets were further pooled over all imputations according to Rubin’s rule [34]. All statistical models were two-sided and based on a significance level of 5% (alpha = 0.05) [35].

To investigate the association between repeat-measured antibodies and clinical outcomes–both measured twice per patient–we created a MLM using the LME4 package v1.1-36 [36]. MLM is superior to the repeated-measures correlation (rmcorr) analysis when dealing with small sample sizes and data models with both random intercepts and slopes [36]. We considered Bonferroni correction for multiple testing [37]. Standardized beta coefficients of all MLM comparisons were visualized through a heatmap plot and use of the GPLOTS package v3.2.0 [38].

We further compared clinical outcomes across three measurement time points with one-way (time) repeated-measures analysis of variance (rmANOVA). Creating linear mixed-effects models (LMM) using the LME4 package v1.1-36, we controlled random subject effects [36]. We used Mauchly’s test of sphericity to test clinical outcome data for normality [35] and visualized data through box plots generated with the GGPLOT2 package v3.5.1 [39]. Concerning the EQ-5D-5L questionnaire we used the eq5d package v0.15.2 to calculate the index score based on a reference German population as there was no Swiss population available [40,41].

3. Results

This pilot study with a statistical power of 10% included seven patients with five being male and two being female. Their mean age was 45 ± 10.13 years with a range between 30 and 57 years.

Due to time constraints, two patients prematurely terminated the study after T1 and T2, lacking two-thirds and one-third of clinical outcome data, respectively. One of these two patients lacked antibody data at the end of therapy. The missing values of these patients were further imputed and included in the analyses. Across all MLM comparisons between antibodies and clinical outcomes, there were two statistically significant associations: Higher concentrations of β2-adrenergic receptor antibodies (R2 =.32, t(504) = -2.46, p = .014*) and M3-muscarinic acetylcholine receptor antibody (R2 = .46, t(110) =-3.17, p = .002**) were both relevant predictors of a lower and therefore worse EQ-5D-5L index score.

However, these results did not remain statistically significant after Bonferroni adjustment for multiple testing. Table 2 shows detailed results of all MLM comparisons between antibodies and clinical outcomes measured at T1 and T3, while Figure 1 illustrates all MLM comparisons using a heatmap plot.

There were no statistically significant improvements in clinical outcomes across T1, T2, and T3. However, we found trends towards improvement of health-related quality of life measured with the index score of the EQ-5D-5L questionnaire. See Table 3 for detailed results of the rmANOVA of clinical outcomes over the course of the study. Figure 2 shows unimputed clinical outcomes over the course of the study, measured at T1, T2, and T3 using box plots.

4. Discussion

4.1. Patient Selection

In this pilot study we focused on the central entity ME/CFS, regardless of the presumed triggering factor. It has been proven that ME/CFS is a constellation of symptoms, a well-defined clinical state that could be evoked by several different catalysts. In this chapter we discuss the choice of therapy in this pilot study, namely the combination of PE followed by low-dose IVIG-administration after each session.

• Reasons for IVIG-administration

IVIG is a highly purified concentrate of immunoglobulins, primarily IgG, derived from the pooled plasma of 1,000 to 100,000 healthy donors. Its composition reflects the diverse immunological exposures and antibody repertoires of the donor population. This gives IVIG its broad functionality and utility in both immunodeficiency and autoimmune conditions.[42] ME/CFS patients could benefit from the so-called anti-idiotype antibodies, which neutralize pathogenic autoantibodies and restore immune balance. Furthermore, there are immunomodulatory effects, IVIG impacts immune regulation by modulating both the innate and adaptive arms of the immune system. It can bind to Fc receptors, interact with complement proteins, and neutralize pro-inflammatory mediators, making it effective in autoimmune and inflammatory diseases.[43]

There are various immunomodulatory effects of intravenous immunoglobulins, that can be summarized as follows:

Inhibition of Monocyte and Macrophage Activation: IVIG reduces the activation of monocytes and macrophages. It induces anti-inflammatory cytokines from innate immune cells. Decreases macrophage responsiveness to interferon, dampening inflammatory responses.[18]

Cytotoxicity to Neutrophils and Eosinophils: IVIG exerts cytotoxic effects on neutrophils and eosinophils. This shifts the immune environment from pro-inflammatory to anti-inflammatory.

Expansion of Regulatory T Cells: IVIG enhances immunosuppressive effects by expanding the population of regulatory T cells, promoting immune tolerance.

Suppression of B-Lymphocytes: IVIG suppresses the proliferation of B lymphocytes, reducing antibody production. It also suppresses autoreactive B lymphocytes and neutralizes their cytokines, mitigating autoimmunity.

Modulation of Endothelial Cells: IVIG influences endothelial cell proliferation and adhesion molecule expression in vascular lumens. This leads to an anti-inflammatory effect, particularly beneficial in vascular disorders.[44]

This combination of mechanisms allows IVIG to effectively modulate cellular immunity and combat a range of autoimmune and inflammatory conditions, furthermore it can address the endothelial dysfunction, which is consumed to play a major role in the pathogenesis of Long-COVID and ME/CFS.

• Plasma exchange for removing micro-clots

The discussion of the major problems was on the one hand the autoantibodies, on the other hand the micro-clots that was found at the Stellenbosch University in South Africa [45]. A summary from 2023 could not prove that the micro-clots are responsible for the symptoms [14]. There again we know that plasma-exchange can improve microcirculation. In this correlation, we decided to perform plasma-exchange, with the hypothesis to put out the micro-clots and the autoantibodies at once.

Other studies observed significant correlations between the levels of most antibodies and key symptoms such as fatigue and muscle pain in patients with an infection-triggered onset. Cognitive impairment severity was associated with AT1-R(angiotensin-1 receptor)-antibody and ETA-R-AAB (Endothelin-1 type A receptor antibody), while gastrointestinal symptom severity correlated with anti-adrenergic α1/2 receptor antibody (alpha1/2-AdR-AAB). In contrast, patients with non-infection-triggered ME/CFS exhibited fewer and distinct correlations [9].

4.2. Data Collection and Patient Compliance

Working with ME/CFS patients can be challenging. The core symptom, the post-exertional malaise (the so-called “crushes”) could interfere with the adequate data collection, even if the study group works with a well-established study protocol. Some of the patients suffered from recurring post-exertional malaise after the conduction of 6MWT. Therefore our study group decided to cease the 6MWT for the wellbeing of our patients. Consequently, the 6MWT dataset was not eligible for statistical analysis. ME/CFS itself, due to its main clinical manifestation characterized by exertion intolerance and the post-exertional malaise poses a huge challenge for adequate data collecting. Sometimes during the crushes, ME/CFS patients have to cancel consultations or a blood test because of the major malaise. This can negatively impact the exact sample collection. One patient had to be excluded because of lacking baseline and follow-up clinical outcome data. 2 patients could be analyzed after data imputation.

4.3. Subjective Experience of Patients

Most patients reported a subjective benefit after the first PE + IVIG therapy, however it could be observed that many of them were crushed after the second round. This deterioration of the clinical status led to premature termination of the therapy in 3 cases. During the 4 treatments most patients experienced a gradual improvement in their symptoms. 5 of a total of 7 patients reported a subjective benefit after the PE therapy.

4.4. Vascular Access

Vascular access is often a subject of discussion related to extracorporeal therapies. Among the seven enclosed patients, five have had peripheral venous access, inserted directly before the PE. Two of them needed a central venous catheter for the PE, one of them was reported to have been infected and therefore must have been prematurely extracted. All therapies have been performed in an outpatient-setting.

5. Conclusions

Our study has identified a significant association between elevated levels of ꞵ2-adrenergic receptor antibodies and M3-receptor antibodies in patients with post-COVID syndrome. The data indicate that higher concentrations of these autoantibodies are associated with a deterioration in quality of life, highlighting their potential role in the pathophysiology of this condition.

Five of seven patients reported an explicit benefit after the treatments. Even though these subjective patient-experiences are not suitable for statistical analysis, they gave us a confirming hint that our statistical evaluations are correlating with the beneficial clinical outcome of our patients.

To mitigate the effects of these autoantibodies, we implemented plasma exchange as a therapeutic intervention, successfully reducing their levels in patients. Following each plasma exchange procedure, we administered 2g of human immunoglobulin (Octagam) to support the immune system and replenish necessary antibodies. This approach not only aims to alleviate symptoms associated with autoantibody-mediated dysfunction but also to provide a protective effect against potential infections.

However, our findings suggest that additional therapeutic strategies may be warranted for these patients. Specifically, B cell depletion therapy could further reduce the production of these harmful autoantibodies and enhance overall treatment efficacy. By integrating plasma exchange, human immunoglobulin administration, and B cell-targeted therapies, we may be able to significantly improve the quality of life for individuals suffering from ME/CFS. Future studies are needed to validate these therapeutic strategies and optimize management approaches for this complex and challenging condition.