Chronic fatigue syndrome: An emerging sequela in COVID-19 survivors?, by Elisa Mantovani, Sara Mariotto, Daniele Gabbiani, Gianluigi Dorelli, Silvia Bozzetti, Angela Federico, Serena Zanzoni, Domenico Girelli, Ernesto Crisafulli, Sergio Ferrari, Stefano Tamburin inJournal of NeuroVirology August 2, 2021
Research abstract:
SARS-CoV-2 survivors may report persistent symptoms that resemble myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS).
We explored (a) ME/CFS-like symptom prevalence and (b) whether axonal, inflammatory, and/or lung changes may contribute to ME/CFS-like symptoms in SARS-CoV-2 survivors through clinical, neuropsychiatric, neuropsychological, lung function assessment, and serum neurofilament light chain, an axonal damage biomarker.
ME/CFS-like features were found in 27% of our sample.
ME/CFS-like group showed worse sleep quality, fatigue, pain, depressive symptoms, subjective cognitive complaints, Borg baseline dyspnea of the 6-min walking test vs. those without ME/CFS-like symptoms. These preliminary findings raise concern on a possible future ME/CFS-like pandemic in SARS-CoV-2 survivors.
This review raises a number of compelling issues related to the condition of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS). Some historical perspective is necessary in order to highlight the nature of the controversy concerning its causation.
Throughout history, a pattern tends to repeat itself when natural phenomena require explanation. Dogma usually arrives first, then it is eventually replaced by scientific understanding.
The same pattern is unfolding in relation to ME/CFS, but supporters of the psychological dogma surrounding its causation remain stubbornly resistant, even in the face of compelling scientific evidence to the contrary. Acceptance of the latter is not just an academic issue; the route to proper understanding and treatment of ME/CFS is through further scientific research rather than psychological theorisation. Only then will a long-suffering patient group benefit.
Extract:
Dr William Weir
In conclusion, proper scientific research into the physical cause(s) of ME/CFS will eventually replace the damaging influence of pseudoscientific, psychological dogma. A reliable biomarker currently in development [25] is a big step in this direction.
Also, the current Covid19 pandemic may be a cloud with a silver lining. “LongCovid”, a devastating aftermath of Covid19 infection, is currently attracting research funding. The clinical presentations of “LongCovid” are strikingly similar to those of ME/CFS, and the underlying pathology may well be the same [26]. Hopefully, the funds referred to will be used for properly directed scientific searches for the precise cause of this pathology, rather than for a PACE mark 2.
To paraphrase Albert Einstein:
“the definition of insanity is to do the same thing again, expecting a different result”.
If sanity prevails, properly focussed scientific research will eventually bring much needed relief to a population of patients who have hitherto been very poorly served by the medical profession.
Clinical similarities between chronic fatigue syndrome and idiopathic intracranial hypertension, supported by measurements of intracranial pressure, invite suggestions that they may be connected, the first representing a mild version of the second. Yet, if this is to be the basis for a structural explanation for chronic fatigue syndrome, it already seems incomplete, failing to explain cases where disability seems disproportionate. Is there some other confounding variable?
Purpose
To refine, in this theoretical paper, an earlier model connecting chronic fatigue syndrome with idiopathic intracranial hypertension to allow for a cerebrospinal fluid (CSF) leak.
Cerebrospinal fluid (CSF) is a clear, colorless body fluid found within the tissue that surrounds the brain and spinal cord of all vertebrates [animals with backbones]. From: Wikipedia
Model
In this model, the primary structural problem is acquired obstruction to cranial venous outflow. This obstruction can take different forms, may be intermittent and subtle, and even be mistaken for normal venous anatomy, yet would be the driving force behind a tendency towards increased intracranial pressure. This chronic elevation of intracranial pressure stresses the dural membrane maintaining the integrity of the subarachnoid space, which can rupture at a weak point, allowing CSF to leak away and intracranial pressure to fall. The clinical manifestation of this disorder is the product of the severity of cranial venous outflow compromise and of the competing forces on intracranial pressure.
Figure 1. Obstruction to cranial venous outflow causes a rise in intracranial venous pressures leading to a rise in intracranial pressure and the syndrome of IIH. If a CSF leak develops before IIH becomes evident then the physiological disturbance manifests as spontaneous intracranial hypotension (SIH). IIH and SIH have multiple overlapping symptoms and patients may reach an equilibrium position between them, or may cycle between one and the other, reflecting opposing forces on intracranial pressure.
In some instances, a CSF leak will mitigate the effects of venous compromise, in others it will compound it, producing a disease spectrum ranging through idiopathic intracranial hypertension, chronic fatigue syndrome, fibromyalgia, and spontaneous intracranial hypotension.
Conclusion
In chronic fatigue syndrome a normal intracranial pressure does not exclude significant physiological disturbance.
Extract from full article:
We suggest, therefore, that cranial venous outflow obstruction can take different forms, may be intermittent and subtle, and even mistaken as part of normal variant anatomy, yet be the driving force behind a disorder of intracranial pressure in which pressure may be high, normal or low, depending on the nature and severity of venous compromise and whether or not it is complicated by a CSF leak.
We suggest that the clinical manifestation of this disorder will reflect, not just intracranial pressure, but also the underlying venous obstruction, and is represented by a spectrum which includes chronic fatigue syndrome, fibromyalgia, spontaneous intracranial hypotension and IIH. We suspect that a similar mechanism may be operative in other medically unexplained syndromes.
Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a complex, multisystem, and profoundly debilitating neuroimmune disease, probably of post-viral multifactorial etiology. Unfortunately, no accurate diagnostic or laboratory tests have been established, nor are any universally effective approved drugs currently available for its treatment.
This study aimed to examine whether oral coenzyme Q10 and NADH (reduced form of nicotinamide adenine dinucleotide) co-supplementation could improve perceived fatigue, unrefreshing sleep, and health-related quality of life in ME/CFS patients.
A 12-week prospective, randomized, double-blind, placebo-controlled trial was conducted in 207 patients with ME/CFS, who were randomly allocated to one of two groups to receive either 200 mg of CoQ10 and 20 mg of NADH (n = 104) or matching placebo (n = 103) once daily. Endpoints were simultaneously evaluated at baseline, and then reassessed at 4- and 8-week treatment visits and four weeks after treatment cessation, using validated patient-reported outcome measures.
A significant reduction in cognitive fatigue perception and overall FIS-40 score (p < 0.001 and p = 0.022, respectively) and an improvement in HRQoL (health-related quality of life (SF-36)) (p < 0.05) from baseline were observed within the experimental group over time. Statistically significant differences were also shown for sleep duration at 4 weeks and habitual sleep efficiency at 8 weeks in follow-up visits from baseline within the experimental group (p = 0.018 and p = 0.038, respectively).
Overall, these findings support the use of CoQ10 plus NADH supplementation as a potentially safe therapeutic option for reducing perceived cognitive fatigue and improving the health-related quality of life in ME/CFS patients.
Future interventions are needed to corroborate these clinical benefits and also explore the underlying pathomechanisms of CoQ10 and NADH administration in ME/CFS.
5. Conclusions
To the best of our knowledge, this is the first study to assess the effects of oral CoQ10 plus NADH supplementation administered to a substantial number of ME/CFS patients (n = 207). Our findings suggest that, over a two-month period, this combination is potentially effective in reducing cognitive fatigue (also known as “brain fog”) and overall fatigue perception, thus improving HRQoL in ME/CFS.
The study shows that CoQ10 and NADH can be safely co-administered to ME/CFS patients and are generally well-tolerated at the dosages indicated. A therapeutic effect was also demonstrated on sleep quality within the experimental group.
Long-term RCTs in larger ME/CFS cohorts should now be performed to confirm the effectiveness of CoQ10 and NADH co-supplementation in treating the hallmark symptom of post-exertional malaise using two-day consecutive cardiopulmonary exercise testing (2-day CPET).
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.
Serology is the scientific study of serum (a component of blood) and other body fluids. (from Wikipedia)
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.
Uploading serum cups
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.
Discussion extracts:
This new finding was only possible due to the stratification of patients according to a question related to the occurrence of an infection at disease onset together with a sensitivity analysis of the seropositivity cutoff used. Patients’ stratification or subtyping was performed in line with past recommendations for ME/CFS research (28). Following this recommendation, we previously performed an immunological investigation based on a stratification of ME/CFS patients according to the severity of their symptoms (32). By using this stratification, we showed perturbations in the T-cell compartment, namely, in effector CD8+ T cells and in the mucosal-associated invariant T cells.
In another study using similar stratification of the samples from the UKMEB, the levels of the cellular stress biomarker GDF15 were found to be increased in severely affected patients at different time points (45). We speculate that other immunological perturbations could be detected if our alternative stratification could have been used. This investigation will be carried out in the near future.
…As final remarks, we should also note that cutoffs for detecting associations between herpesviruses and ME/CFS might vary from one lab to another and with the serological kits used. In addition, a high cutoff for the data might not define seropositivity per se, but rather a high antibody response whose detection could be the primary objective of the analysis (30, 31). The use of a high cutoff is also in accordance with a modeling approach where seropositivity might be subdivided into different levels (60–62).
Therefore, our sensitivity-like approach seems to have the capacity to detect further serological associations beyond the ones based on the classical seroprevalence. Such a capacity could increase the chance of reaching scientific reproducibility. We then recommend the routine use of this approach in future serological investigations of ME/CFS.
There is a lack of research regarding blood tests within individuals with Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) and between patients and healthy controls. We aimed to compare results of routine blood tests between patients and healthy controls.
Data from 149 patients diagnosed with ME/CFS based on clinical and psychiatric evaluation as well as on the DePaul Symptom Questionnaire, and data from 264 healthy controls recruited from blood donors were compared.
One-way ANCOVA was conducted to examine differences between ME/CFS patients and healthy controls, adjusting for age and gender.
Patients had higher sedimentation rate (mean difference: 1.38, 95% CI: 0.045 to 2.714), leukocytes (mean difference: 0.59, 95% CI: 0.248 to 0.932), lymphocytes (mean difference: 0.27, 95% CI: 0.145 to 0.395), neutrophils (mean difference: 0.34, 95% CI: 0.0 89 to 0.591), monocytes (mean difference: 0.34, 95% CI: 0.309 to 0.371), ferritin (mean difference: 28.13, 95% CI: -1.41 to 57.672), vitamin B12 (mean difference: 83.43, 95% CI: 62.89 to 124.211), calcium (mean difference: 0.02, 95% CI: -0.02 to 0.06), alanine transaminase (mean difference: 3.30, 95% CI: -1.37 to -7.971), low-density lipoproteins (mean difference: 0.45, 95% CI: 0.104 to 0.796), and total proteins (mean difference: 1.53, 95% CI: -0.945 to 4.005) than control subjects.
The patients had lower potassium levels (mean difference: 0.11, 95% CI: 0.056 to 0.164), creatinine (mean difference: 2.60, 95% CI: 0.126 to 5.074) and creatine kinase (CK) (mean difference: 37.57, 95% CI: -0.282 to 75.422) compared to the healthy controls.
Lower CK and creatinine levels may suggest muscle damage and metabolic abnormalities in ME/CFS patients.
Conclusions
Results of several routine blood tests of ME/CFS patients differed from those healthy controls. Our findings particularly highlight that decreased creatinine and CK levels may indicate greater muscle damage and metabolic disturbances in ME/CFS patients and is worthy of future studies. This is also true of results that may indicate a possible low-grade inflammation in ME/CFS patients.
Pilot online self-management course for ME/CFS confirmed in North Wales, Aug-Sep 2021
The Health Board has confirmed that there have been enough applicants to make it worthwhile to run the course. A big thank you to all who helped spread the word. They are happy to accept further registrations until the day before the initial session so please continue to let people with ME/CFS know about it, so they can also help to test the course.
live and/or receive healthcare in north Wales (Anglesey, Conwy, Denbighshire, Flintshire, Gwynedd and Wrexham, as well as some parts of Mid Wales)
have internet access and webcam
are willing to learn how to use Microsoft Teams to take part in sessions – help is available
About the course:
A self-management course has been developed by the EPP team for people who are experiencing Post Viral Syndrome (PVS) and they wish to test it with people living with ME/CFS, including those with no identified viral trigger.
The free course will take into account developments in NICE guidance and aims to help people maintain and improve their quality of life by learning important skills, including:
managing your symptoms
dealing with stress, depression and low self-image
managing pain
developing coping skills
learning ways to relax and eat healthily
working more closely with those caring for you
planning for the future.
When?
for 7 weeks beginning 19th August 2021
twice a week: Tuesday and Thursday
between 11 and 12.30, with breaks
applications will be accepted until 18th August
an introduction session will be held prior to to the start to help get you connected
More information about the ME Self-Management Programme (MESMP) :
Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a disease with unknown etiology, no validated specific and sensitive biomarker, and no standard approved effective treatment. ME/CFS has a profound impact on the quality of life of both patients and caregivers and entails high costs for society. The severity varies among patients who are able to participate to some extent in social life (mild), those who are mainly housebound (moderate) or bedridden (severe), and the very severely ill who are completely dependent on assistance for all daily living tasks, such as feeding or turning around in bed.
Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) often starts in previously healthy individuals after an infection, the most common being infectious mononucleosis (EBV). It is more frequent in women and influenced by genetic predisposition. The main symptoms are postexertional malaise (PEM), fatigue, orthostatic intolerance, cognitive disturbances, sleep problems with inadequate restitution after rest, sensory hypersensitivity with pain, and symptoms related to autonomic and immune dysfunction. The prevalence is 0.1% to 0.8%, and ME/CFS must be distinguished from general fatigue, which is much more common in the population.
Historically, there has been limited scientific interest in ME/CFS.
However, research efforts have increased in the last decade. Although this has led to different hypotheses, a firmly established pathomechanism is lacking.
Herein, we suggest a framework model for the initiation and maintenance of ME/CFS consisting of three principal steps:
(a) an initial aberrant immune response;
(b) an effector system for symptom generation and maintenance; and
(c) compensatory adaptations.
The model and possible therapeutic opportunities are summarized in Figure 1.
Conclusions
Our proposed pathomechanistic model is compatible with the lack of obvious histologic inflammation in tissue samples from ME/CFS, lack of overt organ damage, and the potential for recovery — sometimes spontaneous and without sequelae. Future research should focus on the natural course of ME/CFS over time to identify the mechanisms that induce and maintain disease, find targets for intervention, and specifically aim to elucidate immune dysregulation and patterns of autoantibodies with mechanisms for circulatory disturbances.
In our model, clinical symptoms of ME/CFS are related primarily to the inadequate autoregulation of blood flow yielding tissue hypoxia on exertion, but are also influenced by the compensatory adaptations from increased sympathetic output and from metabolic shifts.
We speculate that cognitive techniques, which are reported to help subgroups of patients, might act by modulating the sympathetic output. If so, one would expect a greater benefit for patients with less ongoing immune activation and less vascular dysregulation, but with main symptom contributions from the secondary autonomic adaptations. Conversely, patients with active immune disturbance and ongoing vascular dysregulation as the main symptom generators would have less impact from cognitive intervention, although psychosocial support and coping strategies may still have a beneficial impact on their quality of life.
In conclusion, we suggest that ME/CFS in a subgroup of patients is a variant of an autoimmune disease, with a role for B cells/plasma cells and a pattern of autoantibodies emerging after infection and persisting over time. Key symptoms may result from the consequent functional disturbance in blood flow autoregulation causing tissue hypoxia on exertion and associated autonomic and metabolic responses to maintain energy homeostasis.
Finally, there is growing concern for patients with “long COVID.” Research is needed to determine whether the symptoms, which may resemble those of ME/CFS, are caused by subtle organ damage from the viral infection or whether subgroups of “long haulers” actually have a postinfectious immune disturbance and pathomechanism similar to those in ME/CFS.
Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is an illness defined predominantly by symptoms. Routine laboratory test results often are normal, raising the question of whether there are any underlying objective abnormalities.
In the past 20 years, however, new research technologies have uncovered a series of biological abnormalities in people with ME/CFS. Unfortunately, many physicians remain unaware of this, and some tell patients that “there is nothing wrong” with them. This skepticism delegitimizes, and thereby multiplies, the patients’ suffering.
Excerpt:
A large literature now describes multiple underlying biological abnormalities in people with ME/CFS. Some of the evidence comes from tests that have been available for decades but are not part of the “standard” laboratory test battery [4], and some evidence comes from the new technologies mentioned above. Unfortunately, many physicians are unaware of the new discoveries about ME/CFS.
The abnormalities all converge on and can affect the brain, and fall into five categories.
First, there are anatomic, physiologic and electrical abnormalities in the brain [5].
Second, various elements of the immune system are chronically activated and in some people those elements are exhausted—perhaps secondary to years of chronic activation [5]. This includes chronic activation of the brain’s innate immune system—neuroinflammation [6]. It also includes evidence of autoimmunity, including autoantibodies directed at targets in the central and autonomic nervous system [7].
Third, there also is evidence of impaired energy metabolism: the person with ME/CFS feels he or she lacks “energy” because his or her cells have a reduced ability to generate energy molecules (adenosine triphosphate, or ATP) [8]. Along with the abnormalities in energy metabolism, there is associated oxidative stress, or redox imbalance [8].
Fourth, the autonomic nervous system is dysregulated, one consequence of which appears to be impaired blood flow to the brain [9].
Fifth, there are characteristic abnormalities of the gut microbiome [10], with increased numbers of pro-inflammatory bacterial species and decreased numbers of butyrate-producing anti-inflammatory species.
What remains unclear are the mechanistic details as to how the abnormalities in each of these five categories affect each other, and whether one of them is the initial and primary abnormality [5,8]. In this next decade, the growing community of global investigators who are studying ME/CFS should place a high priority on refining our understanding of each of these categories of abnormality, and an even higher priority on understanding how they are connected. This is essential for developing good diagnostic tests, and effective treatments.
Whitney Dafoe ends the description of his suffering by emphasizing the silver lining around the cloud that he has lived with for nearly 20 years. He says he has learned a great deal about what is important in life, and that “ME/CFS is the greatest teacher I’ve ever had.”
I would like to think that ME/CFS will also prove to be a great teacher to the growing community of physicians and biomedical investigators involved in caring for and studying the illness. In particular, I speculate that the connections between the various abnormalities involving the central and autonomic nervous system, immune system, energy metabolism, redox imbalance, and the human microbiome that have been noted in ME/CFS will prove to be central also to the pathophysiology of many other diseases.
In particular, the COVID-19 pandemic appears to be producing millions of new cases of an ME/CFS-like condition [11], and NIH has allocated more than $1 billion to study this and other post-COVID chronic illnesses. Hopefully, this investment will produce more answers.
Of the personal lessons that I, as a physician, have learned from ME/CFS, perhaps the most important is that, if patients tell you they are suffering, your default assumption should be to believe them—even if you cannot find an answer with the diagnostic technology you first deploy. Above all, never succumb to the temptation to dismiss the patient’s symptoms because you cannot explain them. That may ease your anxiety, but it only multiplies the patient’s suffering.
The neuroprotective ratio kynurenic acid / 3-hydroxykykynurenine is lower in FM compared to healthy controls.
The KAT II enzymatic activity (xanthurenic acid / 3-hydroxykynurenine) is lower in FM compared to healthy controls
BMI, fatigue and pain are related to kynurenine pathway metabolic concentrations.
The metabolic trap hypothesis suggests that a metabolic problem exists in one or more areas of a person with ME/CFS, with a defect in the IDO2 enzyme of the tryptophan kynurenine pathway being identified as a possible metabolic trap. from MEpedia
Research abstract:
Background:
There is growing evidence that the kynurenine pathway is involved in the pathology of diseases related to the central nervous system (CNS), because of the neuroprotective or neurotoxic properties of certain metabolites, yet the role of each metabolite is not clear.
The pathology of Chronic Fatigue Syndrome (CFS) and Fibromyalgia (FM) is currently under investigation, and the overlapping symptoms such as depression suggest that the CNS may be involved. These symptoms may be driven by enhanced neurotoxicity and/or diminished neuroprotection.
However, the kynurenine metabolite status has not been well studied in these two possible related disorders of CFS and FM. The objective of this study was to investigate the metabolites and ratios of the kynurenine pathway in CFS and FM compared to healthy controls and examine the possible correlations with symptoms of anxiety and depression.
Method:
In this study, females aged 18-60 were included: 49 CFS patients; 57 FM patients; and 54 healthy controls. Blood plasma was analysed for the following metabolites involved in the kynurenine pathway: Tryptophan, kynurenine, kynurenic acid (KA), 3-hydroxykykynurenine (HK), anthranilic acid, xanthurenic acid (XA), 3-hydroxyanthranilic acid, quinolinic acid (QA) and picolinic acid.
The concentrations of these metabolites, as well as the ratios of different metabolites indicating enzymatic activity, were compared between the groups. Findings were controlled for age, body mass index (BMI), and symptoms of anxiety and depression.
Results:
QA differed between CFS and FM patients (β = .144, p = .036) and was related to higher levels of BMI (β = .017, p = .002). The neuroprotective ratio given by KA/QA was lower for CFS patients compared to healthy controls (β = -.211, p = .016). The neuroprotective ratio given by KA/HK was lower for FM patients compared to healthy controls, and this lower neuroprotective ratio was associated with increased symptoms of pain. The kynurenine aminotransferase II (KAT II) enzymatic activity given by XA/HK was lower for FM patients compared to healthy controls (β = -.236, p = .013). In addition, BMI was negatively associated with enhanced KAT II enzymatic activity (β = -.015, p = .039). Symptoms of anxiety and depression were not associated with the metabolites or ratios studied.
Conclusion:
Our study indicates associations between kynurenine metabolism and CFS and FM as well as characteristic symptoms like fatigue and pain. Forthcoming studies indicating a causative effect may place kynurenine metabolites as a target for treatment as well as prevention of these conditions in the future.
5. Conclusion
CFS patients may have lower neuroprotection due to higher levels of QA and lower neuroprotective ratio (KA/QA) than healthy controls. Fatigue and pain – central factors in CFS and FM – seem to be particularly related to AA, QA, and KAT II activity. Body weight reduction and smoking cessation may be beneficial in chronic fatigue and pain conditions. Kynurenine metabolites and ratios can be promising indicators and targets of diagnosis and treatment of both FM and CSF. However, caution should be taken because of the complexity of the symptoms in these patients, such as fatigue and pain, and their underlying mechanisms, independent of diagnostic groups
This study aimed to examine whether oral coenzyme Q10 and NADH (reduced form of nicotinamide adenine dinucleotide) co-supplementation could improve perceived fatigue, unrefreshing sleep, and health-related quality of life in ME/CFS patients.
There is a lack of research regarding blood tests within individuals with Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) and between patients and healthy controls. We aimed to compare results of routine blood tests between patients and healthy controls.
A self-management course has been developed by the 
There is growing evidence that the 
