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Most patients with Post COVID Syndrome (PCS) present with a plethora of symptoms without clear evidence of organ dysfunction. A subset of them fulfills diagnostic criteria of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). Symptom severity of ME/CFS correlates with natural regulatory autoantibody (AAB) levels targeting several G-protein coupled receptors (GPCR). In this exploratory study, we analyzed serum AAB levels against vaso- and immunoregulatory receptors, mostly GPCRs, in 80 PCS patients following mild-to-moderate COVID-19, with 40 of them fulfilling diagnostic criteria of ME/CFS. Healthy seronegative (n=38) and asymptomatic post COVID-19 controls (n=40) were also included in the study as control groups. We found lower levels for various AABs in PCS compared to at least one control group, accompanied by alterations in the correlations among AABs. Classification using random forest indicated AABs targeting ADRB2, STAB1, and ADRA2A as the strongest classifiers (AABs stratifying patients according to disease outcomes) of post COVID-19 outcomes. Several AABs correlated with symptom severity in PCS groups. Remarkably, severity of fatigue and vasomotor symptoms were associated with ADRB2 AAB levels in PCS/ME/CFS patients. Our study identified dysregulation of AAB against various receptors involved in the autonomous nervous system (ANS), vaso-, and immunoregulation and their correlation with symptom severity, pointing to their role in the pathogenesis of PCS.

Infections by the Epstein-Barr virus (EBV) are often at the disease onset of patients suffering from Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS). However, serological analyses of these infections remain inconclusive when comparing patients with healthy controls (HCs). In particular, it is unclear if certain EBV-derived antigens eliciting antibody responses have a biomarker potential for disease diagnosis. With this purpose, we re-analyzed a previously published microarray data on the IgG antibody responses against 3,054 EBV-related antigens in 92 patients with ME/CFS and 50 HCs. This re-analysis consisted of constructing different regression models for binary outcomes with the ability to classify patients and HCs. In these models, we tested for a possible interaction of different antibodies with age and gender. When analyzing the whole data set, there were no antibody responses that could distinguish patients from healthy controls. A similar finding was obtained when comparing patients with non-infectious or unknown disease trigger with healthy controls. However, when data analysis was restricted to the comparison between HCs and patients with a putative infection at their disease onset, we could identify stronger antibody responses against two candidate antigens (EBNA4_0529 and EBNA6_0070). Using antibody responses to these two antigens together with age and gender, the final classification model had an estimated sensitivity and specificity of 0.833 and 0.720, respectively. This reliable case-control discrimination suggested the use of the antibody levels related to these candidate viral epitopes as biomarkers for disease diagnosis in this subgroup of patients. To confirm this finding, a follow-up study will be conducted in a separate cohort of patients.

We here provide an overview of the pathophysiological mechanisms during heat stroke and describe similar mechanisms found in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). Both conditions are characterized by disturbed homeostasis in which inflammatory pathways play a central role. Splanchnic vasoconstriction, increased gut permeability, gut-related endotoxemia, systemic inflammatory response, central nervous system dysfunction, blood coagulation disorder, endothelial-cell injury, and mitochondrial dysfunction underlie heat stroke. These mechanisms have also been documented in ME/CFS. Moreover, initial transcriptomic studies suggest that similar gene expressions are altered in both heat stroke and ME/CFS. Finally, some predisposing factors for heat stroke, such as pre-existing inflammation or infection, overlap with those for ME/CFS. Notwithstanding important differences - and despite heat stroke being an acute condition - the overlaps between heat stroke and ME/CFS suggest common pathways in the physiological responses to very different forms of stressors, which are manifested in different clinical outcomes. The human studies and animal models of heat stroke provide an explanation for the self-perpetuation of homeostatic imbalance centered around intestinal wall injury, which could also inform the understanding of ME/CFS. Moreover, the studies of novel therapeutics for heat stroke might provide new avenues for the treatment of ME/CFS. Future research should be conducted to investigate the similarities between heat stroke and ME/CFS to help identify the potential treatments for ME/CFS.

The evidence of an association between Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) and chronic herpesviruses infections remains inconclusive. Two reasons for the lack of consistent evidence are the large heterogeneity of the patients' population with different disease triggers and the use of arbitrary cutoffs for defining seropositivity. In this work we re-analyzed previously published serological data related to 7 herpesvirus antigens. Patients with ME/CFS were subdivided into four subgroups related to the disease triggers: S0-42 patients who did not know their disease trigger; S1-43 patients who reported a non-infection trigger; S2-93 patients who reported an infection trigger, but that infection was not confirmed by a lab test; and S3-48 patients who reported an infection trigger and that infection was confirmed by a lab test. In accordance with a sensitivity analysis, the data were compared to those from 99 healthy controls allowing the seropositivity cutoffs to vary within a wide range of possible values. We found a negative association between S1 and seropositivity to Epstein-Barr virus (VCA and EBNA1 antigens) and Varicella-Zoster virus using specific seropositivity cutoff. However, this association was not significant when controlling for multiple testing. We also found that S3 had a lower seroprevalence to the human cytomegalovirus when compared to healthy controls for all cutoffs used for seropositivity and after adjusting for multiple testing using the Benjamini-Hochberg procedure. However, this association did not reach statistical significance when using Benjamini-Yekutieli procedure. In summary, herpesviruses serology could distinguish subgroups of ME/CFS patients according to their disease trigger, but this finding could be eventually affected by the problem of multiple testing.

Soluble cluster of differentiation 26 (sCD26) has a wide range of enzymatic functions affecting immunological, metabolic and vascular regulation. Diminished sCD26 concentrations have been reported in various autoimmune diseases and also in Myalgic Encephalomyelitis/Chronic fatigue syndrome (ME/CFS). Here we re-evaluate sCD26 as a diagnostic marker and perform a comprehensive correlation analysis of sCD26 concentrations with clinical and paraclinical parameters in ME/CFS patients. Though this study did find significantly lower concentrations of sCD26 only in the female cohort and could not confirm diagnostic suitability, results from correlation analyses provide striking pathomechanistic insights. In patients with infection-triggered onset, the associations of low sCD26 with elevated autoantibodies (AAB) against alpha1 adrenergic (AR) and M3 muscarinic acetylcholine receptors (mAChR) point to a pathomechanism of infection-triggered autoimmune-mediated vascular and immunological dysregulation. sCD26 concentrations in infection-triggered ME/CFS were found to be associated with activated T cells, liver enzymes, creatin kinase (CK) and lactate dehydrogenase (LDH) and inversely with Interleukin-1 beta (IL-1b). Most associations are in line with the known effects of sCD26/DPP-4 inhibition. Remarkably, in non-infection-triggered ME/CFS lower sCD26 in patients with higher heart rate after orthostatic challenge and postural orthostatic tachycardia syndrome (POTS) suggest an association with orthostatic regulation. These findings provide evidence that the key enzyme sCD26 is linked to immunological alterations in infection-triggered ME/CFS and delineate a different pathomechanism in the non-infectious ME/CFS subset.

Primaquine (PQ) is an antimalarial drug with the potential to reduce malaria transmission due to its capacity to clear mature Plasmodium falciparum gametocytes in the human host. However, the large-scale roll-out of PQ has to be counterbalanced by the additional risk of drug-induced hemolysis in individuals suffering from Glucose-6-phospate dehydrogenase (G6PD) deficiency, a genetic condition determined by polymorphisms on the X-linked G6PD gene. Most studies on G6PD deficiency and PQ-associated hemolysis focused on the G6PD A- variant, a combination of the two single nucleotide changes G202A (rs1050828) and A376G (rs1050829), although other polymorphisms may play a role. In this study, we tested the association of 20 G6PD single nucleotide polymorphisms (SNPs) with hemolysis measured seven days after low single dose of PQ given at the dose of 0.1 mg/kg to 0.75 mg/kg in 957 individuals from 6 previously published clinical trials investigating the safety and efficacy of this drug spanning five African countries. After adjusting for inter-study effects, age, gender, baseline hemoglobin level, PQ dose, and parasitemia at screening, our analysis showed putative association signals from the common G6PD mutation, A376G [−log10(p-value) = 2.44] and two less-known SNPs, rs2230037 [−log10(p-value] = 2.60), and rs28470352 [−log10(p-value) = 2.15]; A376G and rs2230037 were in very strong linkage disequilibrium with each other (R2 = 0.978). However, when the effects of these SNPs were included in the same regression model, the subsequent associations were in the borderline of statistical significance. In conclusion, whilst a role for the A- variant is well established, we did not observe an important additional role for other G6PD polymorphisms in determining post-treatment hemolysis in individuals treated with low single-dose PQ.

Background: Antimalarial antibody measurements are useful because they reflect historical and recent exposure to malaria. As such, they may provide additional information to assess ongoing transmission in low endemic or pre-elimination settings where cases are rare. In addition, the absence of antibody responses in certain individuals can indicate the cessation of transmission. Commercial malaria enzyme-linked immunosorbent assays (ELISA) detect antimalarial antibodies and are commonly used to screen blood donations for possible malaria infection. However, there is no standardized test to detect antimalarial antibodies for epidemiological use. Here we compared five commercially available ELISA kits (Trinity Biotech, newbio, DiaPro, Cellabs, and NovaTec) in search of a standardized tool for supporting claims of absence of malaria transmission. For comparison, a research-based (RB) ELISA protocol was performed alongside the commercial kits.Results: The commercial kits were first compared using serum samples from known malaria-unexposed individuals (n = 223) and Toxoplasma-infected individuals (n = 191) to assess specificity and cross-reactivity against non-malaria infections. In addition, 134 samples from ≥10-year-olds collected in a hyperendemic region in the Gambia in the early 1990s were used to assess sensitivity. Three out of five kits showed high sensitivity (90–92%), high specificity (98–99%), low cross-reactivity (0–3%) and were considered user-friendly (Trinity Biotech, newbio and NovaTec). Two of these kits (Trinity Biotech and NovaTec) were taken forward for epidemiological evaluation and results were compared to those using the RB-ELISA. Samples from two pre-elimination settings (Praia, Cape Verde; n = 1,396, and Bataan, the Philippines; n = 1,824) were tested. Serological results from both the Trinity Biotech kit and the RB-ELISA concurred with recent passively detected case counts in both settings. Results from the Trinity Biotech kit reflected a significant decrease in the number of reported cases in Bataan in the 1990s better than the RB-ELISA. Results from the NovaTec kit did not reflect transmission patterns in either setting.Conclusion: The Trinity Biotech commercial ELISA kit was considered reliable for epidemiological use and accurately described transmission patterns in two (previously) malaria endemic settings. The use of this simple and standardized serological tool may aid national control and elimination programs by confirming that regions are free from malaria.

Autoimmunity and chronic viral infections are recurrent clinical observations in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS), a complex disease with an unknown cause. Given these observations, the regulatory CD4+ T cells (Tregs) show promise to be good candidates for the underlying pathology due to their capacity to suppress the immune responses against both self and microbial antigens. Here, we discussed the overlooked role of these cells in the chronicity of Human Herpes Virus 6 (HHV6), Herpes Simplex 1 (HSV1), and Epstein–Barr virus (EBV), as often reported as triggers of ME/CFS. Using simulations of the cross-regulation model for the dynamics of Tregs, we illustrated that mild infections might lead to a chronically activated immune responses under control of Tregs if the responding clone has a high autoimmune potential. Such infections promote persistent inflammation and possibly fatigue. We then hypothesized that ME/CFS is a condition characterized by a predominance of this type of infections under control of Tregs. In contrast, healthy individuals are hypothesized to trigger immune responses of a virus-specific clone with a low autoimmune potential. According to this hypothesis, simple model simulations of the CD4+ T-cell repertoire could reproduce the increased density and percentages of Tregs observed in patients suffering from the disease, when compared to healthy controls. A deeper analysis of Tregs in the pathogenesis of ME/CFS will help to assess the validity of this hypothesis.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a debilitating condition with unknown aetiology, Myalgic encephalomyelitis unclear pathophysiology and with no diagnostic test or biomarker available. Many patients report their ME/CFS began after an acute infection, and subsequent increased frequency of infections, such as colds or influenza, is common. These factors imply an altered immunological status exists in ME/CFS, in at least a proportion of patients, yet previous studies of peripheral immunity have been discrepant and inconclusive. The UK ME/CFS Biobank, which has collected blood samples from nearly 300 clinically-confirmed ME/CFS patients, enables large-scale studies of immunological function in phenotypically well-characterised participants. In this study, herpes virus serological status and T cell, B cell, NK cell and monocyte populations were investigated in 251 ME/CFS patients, including 54 who were severely affected, and compared with those from 107 healthy participants and with 46 patients with Multiple Sclerosis. There were no differences in seroprevalence for six human herpes viruses between ME/CFS and healthy controls, although seroprevalence for the Epstein-Barr virus was higher in multiple sclerosis patients. Contrary to previous reports, no significant differences were observed in NK cell numbers, subtype proportions or in vitro responsiveness between ME/CFS patients and healthy control participants. In contrast, the T cell compartment was altered in ME/CFS, with increased proportions of effector memory CD8+ T cells and decreased proportions of terminally differentiated effector CD8+ T cells. Conversely, there was a significantly increased proportion of mucosal associated invariant T cells (MAIT) cells, especially in severely affected ME/CFS patients. These abnormalities demonstrate that an altered immunological state does exist in ME/CFS, particularly in severely affected people. This may simply reflect ongoing or recent infection, or may indicate future increased susceptibility to infection. Longitudinal studies of ME/CFS patients are needed to help to determine cause and effect and thus any potential benefits of immuno-modulatory treatments for ME/CFS.