NIH ME/CFS Deep Phenotyping Study: Part 2. Methods

The US National Institutes of Health (NIH) intramural study on ME/CFS is a three-phase initiative to explore post-infectious ME/CFS in depth, and potentially to identify biomarkers and find treatments. The study was launched in 2016, and the first part, involving more than 70 researchers from a wide range of specialties, has just been completed after almost eight years.

There is a lot of information to digest, so we have split our article into four parts.

1. Overview | 2. Methods | 3. Results | 4. Discussion

What did the study do?

The objective of the study was to utilise “modern broad and deep scientific measures” to describe the biological characteristics of individuals with post-infectious ME/CFS (abbreviated as PI-ME/CFS in the paper) compared to healthy controls. By doing so, the researchers intended to identify differences between the groups that could generate new hypotheses about the pathogenesis (process of disease development) of ME/CFS, and guide future research.

As mentioned in the general overview, the four aims outlined when launching the initiative were to:

Define the clinical phenotype.
Determine the underlying physiology of fatigue (pre and post exercise).
Determine if there are abnormal immune and microbiome profiles.
Determine if features can be reproduced in ex vivo studies
(it is not clear to what extent this aim was addressed).

Strict exclusion criteria resulting in small study population

All participants met the Fukuda criteria, Canadian Consensus Criteria (CCC), or Institute of Medicine (IOM) criteria. Additionally, it was mandatory for individuals with ME/CFS to have “persistent severe fatigue” and post-exertional malaise (PEM) as a consequence of acute infection within the last five years “without a prior history of explanatory, medical or psychiatric illness”.

There were almost 500 initial inquiries about participation for the ME/CFS cohort. However, through application of stringent exclusion criteria in a multi-step selection process (telephone interviews and medical assessment), the number of participants was narrowed down to 17 (accounting for 13 ME/CFS participants who dropped out during the process). Also, there were 21 healthy controls matched for age and body mass index.

Importantly, the study excluded individuals with certain medical conditions (e.g. heart disease), which would make the study procedures, such as exercise tests, risky for the participant. Aside from this, strict exclusion criteria can increase sample homogeneity (uniformity/similarity of characteristics within a population), reducing the potential for confounding variables and allowing for clearer interpretation of results. However, in an attempt to ensure a homogeneous sample, the researchers ended up with a small sample size, which has the opposite effect – impacting generalisability of study findings and reducing the statistical power (the probability that a study will correctly detect a true effect or difference when it exists). Additionally, the number of participants varied across individual assessments; for example, cardiopulmonary exercise testing (CPET) only involved eight ME/CFS participants and nine healthy controls – an incredibly small sample size.

Furthermore, it would have been helpful if the researchers provided clear justification within the paper as to why ME/CFS onset needed to be within five years. This criterion excluded 176 individuals in the initial screening process (although it must be acknowledged that individuals may be excluded from participating for more than one reason).

It is stated that additional recruitment was terminated due to the COVID-19 (SARS-CoV-2) pandemic.

Multidimensional assessment

Participants underwent multidimensional assessment involving evaluation of physiological measures, and physical and cognitive performance, and extensive laboratory testing was conducted on biological samples.

The evaluations conducted are detailed below.

Life narratives and qualitative interviews

“Detailed life narratives and qualitative interviews were conducted to understand the lived experience of PI-ME/CFS, the context of the life it occurred in, and to capture the point-in-time experience of post-exertional malaise.”

It should be noted that two-day cardiopulmonary exercise testing (CPET) – the main method for testing PEM – was not conducted.

Patient Reported Outcomes Measures

At least 12 symptom and health questionnaires were used to collect information about participants in relation to health-related quality of life and nature of symptoms from physical, social and emotional perspectives.

Several of these questionnaires were psychology-centred, including:

The Fatigue Catastrophizing Scale – measures fatigue catastrophising which according to one study is defined as “negative thinking and evaluative patterns toward experienced fatigue”.
Patient Health Questionnaire-15 (PHQ-15) – screens for somatisation (tendency to experience and express psychological distress through physical symptoms).
Belief about Emotions scale – Assesses beliefs about the unacceptability of experiencing or expressing negative emotions.

Psychiatric and Psychological Assessments

At least nine psychiatric/psychological surveys were administered, largely related to depression, anxiety and trauma.

Performance Validity Testing

Performance validity testing is an assessment of whether individuals are genuinely doing their best in neuropsychological tasks, ensuring results are accurate and not unduly influenced by factors such as “poor engagement in testing”. Examples include tests which needed to be done quickly – sorting letters and counting dots – and tests involving remembering words.

Neuropsychological Measures

Neuropsychology is a branch of psychology that studies how a person’s cognition is related to the brain and nervous system. The were 15 neuropsychological tests administered assessing cognitive domains such as memory, attention and processing speed, through various tasks (e.g. word recall, symbol recognition and pattern learning). Additionally, subjective experiences related to effort, performance, and mental and physical fatigue were evaluated.

Notably, within this category is the Effort-Expenditure for Rewards Task (EEfRT) – “a multi-trial game in which participants were offered a choice between two task difficulty levels for a reward”. As per study, “The primary measure of the EEfRT task is Proportion of Hard Task Choices (effort preference). This behavioral measure is the ratio of the number of times the hard task was selected compared to the number of times the easy task was selected.” In this study, EEfRT involved choosing between an easier and harder task involving button-pressing.

Neurological Measures

Brain Imaging: High-resolution brain scans (brain MRI) were conducted to assess the structure and function of the brain.
Neurotransmitter levels: Catecholamines (dopamine, norepinephrine, and related substances) were measured in cerebrospinal fluid (CSF). Catecholamines function as neurotransmitters and hormones within the body.
Measures of brain injury: Plasma (blood) and CSF were analysed for measures of brain injury using immunoassay (a test which utilises antibody-antigen reactions for detecting the presence of a substance).
Small fibre density measures: Skin biopsies and immunostaining (antibody-based technique used to detect and highlight specific proteins/molecules within cells or tissues) were employed to assess for small fibre sensory neuropathy (nerve damage).

Grip Strength and “Physical Fatigue” Assessments

Grip strength was measured through participants squeezing a hand-held device at maximum force for certain amounts of time. “Physical fatigue” was assessed by participants attempting to maintain grip strength at 50% of maximum voluntary contraction during successive time blocks with rest intervals, alongside electromyography (EMG) and transcranial magnetic stimulation (TMS) to measure muscle activity and brain activity. Additionally, functional MRI (brain imaging) was used to monitor brain activity during grip tasks.

Physiological (including Autonomic) Testing

Comprehensive physiological assessments included sleep studies, heart rate monitoring and tilt-table testing to evaluate how the cardiovascular system responds to changes in posture (orthostatic challenge).

One-day CPET was conducted using a cycle ergometer (exercise bike) equipped with a device assessing the physiological response to exercise. Assessments included peak oxygen consumption (VO2), anaerobic threshold and heart rate response. One day prior to CPET, participants stayed within a metabolic chamber (enclosed environment to monitor metabolism) to obtain measures of “energy expenditure and respiratory exchange”.

Analysis of Biological Samples – blood, CSF, stool, muscle

Microbiome Analysis: Stool samples were analysed for microbial composition using genetic sequencing methods. Additionally, the analytical technique, nuclear magnetic resonance (NMR) spectroscopy, was used to study the chemical composition of stool samples.
Immunological Testing: Different methods were employed to analyse components of the immune system, primarily in blood and CSF. This included flow cytometry (laser-based technique) to profile immune cell populations, and a specialised assay to detect autoantibodies.
Mitochondrial testing: Mitochondrial function was evaluated in white blood cells, known as peripheral blood mononuclear cells (PBMCs). Mitochondrial genetic analysis was performed on muscle samples.
Genetic Testing: Further genetic analysis involved RNA sequencing (a technique to assess gene expression), and identification of differentially expressed genes in PBMCs and muscle samples.
Protein Analysis: Blood and CSF were analysed to quantify proteins, including cytokines (signalling proteins) and hormones.

Further laboratory investigations were conducted involving comprehensive analysis of blood and CSF; e.g. complete blood count, inflammatory markers, markers of organ function, metabolites, and certain viral pathogens. Additionally, saliva was tested for cortisol and urine was tested for heavy metals.

Although a wide range of assessments were conducted, certain areas of the study lacked comprehensive testing compared with previous research efforts. For instance, Dysautonomia International, a non-profit organisation dedicated to raising funds for research and promoting awareness of autonomic nervous system disorders, expressed concerns about the autonomic testing and skin biopsy assessments missing key elements from standard testing protocols.