Participants and setting

Participants in this study included 45 patients (40 females and 5 males: age 19–66 years) diagnosed with chronic fatigue syndrome at the CFS Discovery Clinic, a primary care community clinic that works exclusively with CFS/ME patients, located in Donvale (Victoria, Australia). Patients were diagnosed with CFS/ME if they fulfilled the Canadian Diagnostic Criteria, [3] as conducted by a medical practitioner and registered nurse both with 15–20 years’ experience with CFS/ME patients. For the CFS/ME cohort, length of illness ranged from 2 to 40 years. During the early phase of the study, the 2011 International Criteria for CFS/ME diagnosis were published, [4] but since the study had commenced participant recruitment and assessment under the 2003 criteria, the application of the 2003 criteria persisted for the duration of this study.

The study also recruited 17 healthy control participants, comprising 13 females and 4 males (age 24–60 years). Control participants were recruited via neighbouring clinics within the Doncaster district of East Melbourne, through CFS support groups and by word-of-mouth.

Research participants for this study were recruited over 2011–2012 from patients attending CFS Discovery for the first time, often after long periods of interaction with the medical profession, but no conclusive diagnosis or successful treatment. While there are theories, the cause and/or exposure that results in CFS/ME symptoms are not known. A cross-sectional (observation) study design was applied, with CFS/ME cases and healthy controls attending only once for research involvement over the 2011–2012 study period. Follow-up was provided as routine clinic attendance after the research interaction.

Study details

The assessment of patients at CFS Discovery was previously reported by Reynolds et al. [11] which included the protocol for orthostatic intolerance testing and results for autonomic markers of cardiac and circulatory function in CFS/ME patients, with key pathology and cytokine markers for this cohort summarised in Table 1. In addition to the formal CFS/ME diagnostic criteria, assessed by survey and physical examination, research participants were also screened for potentially confounding comorbidities. The study contained tests and clinical assessments to exclude hypothyroidism, Lupus, Fibromyalgia (FM) and Multiple Chemical Sensitivity via the physical and mental status examinations. Also, conducted on each CFS/ME and control participant were routine blood and urine tests, as described below and Table 1. Assessments of sleep patterns were ascertained via the Epworth Scale, and the DASS-36 scale applied to assess anxiety, depression and generalised stress. Recommended guidelines for CFS research also were consulted for this investigation [12], allowing consistent comparisons to other studies. The “Minimal Requirements” under these guidelines were generally captured by the application of the Canadian Criteria to CFS/ME status.

Table 1 CFS/ME and healthy study control participant profile comparisons via representative pathology blood markers and cytokines Full size table

A patient was classified as having CFS/ME if she or he met the above criteria and all other possibilities were excluded as listed above. If a patient’s fatigue was not severe enough, or if the symptom criteria for CFS/ME were not met, he or she was classified as having idiopathic chronic fatigue. Seventeen healthy controls were also recruited and assessed exactly as described for CFS/ME participants, including orthostatic intolerance testing to screen for autonomic comorbidities. Healthy participants could not be a relative of a previous or current CFS/ME patient, or live at the same residential address.

All research participants were non-identifiable to the researchers, with individual identifiers removed from the data provided, except for a unique identification (ID) code. Because only ID codes were provided, no clinical details were attached to samples for activin/follistatin, cytokines and pathology testing, removing potential bias from test result reporting.

As a first attempt at a cross-sectional study on this CFS/ME cohort, a minimum of 30 participants for each group was planned for statistical robustness, and was achieved for the CFS/ME group. Only 17 healthy control participants were recruited within the designated study period, in spite of proactive study advertising and connection with other GP clinics in the district. In addition to the 17 recruited study controls, comparisons of serum activin/follistatin levels with a previously recruited healthy reference population (n = 141) [8] were also conducted for validation.

Patient involvement

No patients were involved in the conception or design of this study, nor were patients asked for input at any stage of the project. A summary of project results is available to research patients, as stated in the participant information pack, on request.

Activin and follistatin analyses

Non-fasting blood samples were collected from patients after the 20-min standing test and the concentrations of activin A, activin B and follistatin were measured in the serum isolated from these samples. Concentrations of activin A were determined using a two-site ELISA (using antibodies supplied by Oxford Brookes University) as previously published [13]. This assay measures both free and follistatin-bound activin A dimers and has no significant cross-reaction with other activin isoforms, such as activin B. Activin B was measured by two-site ELISA, as previously described (using antibodies supplied by Oxford Brookes University) [14]. Follistatin concentrations were determined using an extensively validated radioimmunoassay [15]. Mean activin/follistatin concentrations were compared between the CFS/ME and control cohorts recruited for this study. Further analyses were performed comparing the recruited CFS/ME cohort to data previously collected to calculate reference intervals for each marker.

Pathology and cytokine analyses

In addition to the blood samples collected for activin/follistatin testing, blood was also drawn for pathology testing at Australian Clinical Laboratories (Clayton, Victoria). Tests performed on each CFS/ME and control participant were routine full (complete) blood count examinations, multiple biochemical analyses that included liver and kidney function tests (with 24-h urine analysis), vitamin D (25-OH), parathyroid hormone (PTH), dehydroepiandrosterone sulfate (DHEAS), immunoglobulin (Ig) E, antinuclear antibodies (ANA) and thyroid stimulating hormone (TSH). A fasting glucose tolerance test (GTT) was performed on a separate day post clinic consultation with blood glucose and insulin measured before glucose load (75 g in 200 mL), then at 30 and 60 min post glucose load.

Chronic fatigue syndrome/myalgic encephalomyelitis and control sera were also investigated for interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-17A (IL-17A), tumour necrosis factor (TNF) and interferon-gamma (IFN-γ). The seven cytokines were simultaneously quantitated via multiplex cytometric bead array (human CBA kit, catalogue number 560484, BD Biosciences, North Ryde, NSW), with data acquisition and analysis conducted on a BD FACS Calibur (JCSMR FACS Unit, ANU, Canberra).

Statistical analyses

Comparisons of means or medians between CFS/ME and control groups were performed by independent t test or Mann–Whitney tests respectively, depending on data distribution within the sample and whether the data were continuous or ordinal, with statistical significance accepted at p < 0.05. Whether data followed a normal distribution, or not, was determined by a one-sample Kolmogorov–Smirnov (K–S) Test (SPSS version 22). Data are presented as mean ± SEM.

Chi square (χ2) was calculated to determine the statistical significance (p < 0.05) of the frequency of elevated anti-nuclear antibodies (ANA ≥ 160) between CFS/ME and study control cohorts. Receiver operating curves (ROC) were constructed using SPSS (version 22.0) software, and calculated the area under curve (AUC ± SEM) to distinguish CFS/ME from healthy control cases, using a non-parametric model.

Pathology and activin/follistatin results had no missing data. Missing data was common for the seven serum cytokines measured, and recorded as not detected (ND). Cases with missing data were excluded from the analyses. Mean ± SEM were not reported for TNF and IL-17A since protein was detected in only 2–3 samples across the CFS/ME and control cohorts.

Study approval

All research participants included in this study provided full, signed consent as dictated by the guidelines of the ANU Human Research Ethics Committee (ANU-HREC). This study and associated protocols were conducted after approval by the ANU-HREC (HREC identification 2011/031).