Aim

The aim of this paper is to introduce the weighted standing time (WST) as a proxy for ME/CFS severity, involving an Australian cohort recruited from the greater Melbourne region. Questions addressed to achieve this aim comprise the distribution of WST for healthy controls and patients with ME/CFS, the relationship between WST and Canadian criteria for ME/CFS diagnosis, and the relationship between WST and serum/urine analytes collected both for pathology testing required for screening and monitoring patients, as well as serum cytokine profiles.

Patient cohort

Participants were referred to, or voluntarily presented to, the CFS Discovery Clinic (Donvale, Victoria) between 2011 and 2013. Patients were included if they fulfilled the Canadian Diagnostic ME/CFS criteria [1], and consented to physical examination and biochemical evaluation. Forty-seven consecutive ME/CFS participants were recruited to the study, with two female volunteers excluded from the final cohort due to exceeding the age criteria. One of the remaining 45 patients had a missing standing time measurement (mins), which was estimated using median imputation, allowing the participant to continue in the study. There were 17 control volunteers recruited from the same area of Melbourne, who were in general good health, but not residing with a current patient, nor with a ME/CFS family history. The age range for ME/CFS and healthy control participants was 18–65 years, with a female to male ratio of approximately 4:1.

Inventories

All research participants (ME/CFS and healthy study controls) completed a full history, physical examination and survey assessment to ascertain their profile under the Canadian Criteria for ME/CFS diagnosis. This generated 48 binary variables across neurological, gut, immune activity, pain and so on, as prescribed by the Canadian Consensus Criteria [1], with each criterion scored as 0 or 1 (absent or present) from the clinical assessment, or questionnaire responses. This study commenced just prior to the publication of the International Criteria [18]; therefore, it continued with diagnosis by the Canadian Criteria for the project duration.

As a minimum diagnostic requirement of ME/CFS, activity limiting fatigue must have been present for at least 6 months, and must have featured post-exertional fatigue (“payback”). A comprehensive profile of additional criteria symptoms was explored, including cognitive and neurological function, immune function, sleep, gastrointestinal function, pain and autonomic responses. Screening symptoms and related physical signs of significance are presented by system in Additional file 1: Table S1. In addition to Canadian Criteria screening, all participants were asked to complete a Depression, Anxiety and Stress Scale (DASS) [19]. No participants were excluded due to DASS results.

Weighted standing time

The CFS Discovery orthostatic intolerance (standing test) protocol is described in detail elsewhere [10]. Briefly, participants were required to stand, unaided for a maximum of 20 min after a period of repose necessary for baseline (pre-standing) measurements. Heart rate, blood pressure and oxygen saturations were measured at baseline, and subsequently every 2 min during standing. Parameters were measured at the end of the task (either capped at 20 min, or when the participant could no longer continue) and after 3 min of rest following task completion. A difficulty score was also recorded by the nurse, a subjective measure of how difficult the patient found the standing test. A score between 0 and 10 was recorded (0 = no difficulty standing, 10 = support required to stand, pre-syncope). For this study, two further scores were added, with a subjective score of 12 indicating standing difficulty to the point that the standing test was terminated at less than 20 min (but greater than 10 min), and a score of 14 represented the most extreme difficulty where standing was only possible for 10 min, or less.

With the majority of the ME/CFS cohort achieving a standing time of 20 min, comparisons of standing times for ME/CFS and healthy control cohorts were not informative. To weight the standing time in relation to subjective standing difficulty, and produce a single fatigue response variable, the time standing (maximum 20 min, measured at 2 min intervals) and standing difficulty were combined to produce one measure called the “Weighted Standing Time” (WST). The WST (minutes) was calculated by the following equation:

$${\text{Weighted standing time (WST}}) \, = {\text{ Time standing (mins}}) \times (1 - ({\text{Difficulty}}/14)).$$

WST was calculated for each ME/CFS and study control participant, and the WST score (minutes) used thereafter as a measurable proxy for fatigue severity. For statistical analyses, WST was assigned as the response (dependent) variable for comparisons of blood, urine and cytokines.

Statistical analysis

(i) Clinical (Canadian) criteria—Factor analysis of a tetrachoric correlation matrix (varimax rotation) was conducted to identify significant clinical factors under these diagnostic criteria, as represented by the percentage variance explained by the factor loadings. Tetrachoric correlation allows the analysis of data containing categorical measures, for example “yes” or “no” to the presence of diagnostic symptom under the Canadian Criteria. Analysis of variance was used to determine the statistical difference in factor scores across the WST classes (Table 1).

Table 1 Symptoms and related physical signs based on the Canadian Criteria for ME/CFS diagnosis, presented by class, and as assessed at the CFS Discovery Clinic Full size table

Factor analyses (FA) were performed using the psych() and lattice() libraries of R3.3.0 [20,21,22], applying the FA method described by Tabachnick and Fidell [23]. The individual criteria of “Fatigue ≥ 6 months”, “Limited Activity” and “Payback” were omitted from the factor analysis due to 100% of the patients and 0% of the healthy controls reporting their presence. Details of the factors and their loadings are included in Additional file 1: Tables S2–S10.

(ii) Analytes—For the range of blood, urine and cytokine markers compared for ME/CFS versus study (healthy) controls, descriptive statistics were expressed as mean (± standard deviation) for continuous variables, and as proportions (%) for categorical variables. Two sample tests (Mann–Whitney U) were used to determine whether study (healthy) control and ME/CFS participants could be separated statistically at p < 0.05.

(iii) WST severity scale—The WST was used to define three categories of severity for ME/CFS: mild, moderate and severe. Together with the healthy study controls as a distinct WST category, analysis of variance (ANOVA) and follow-up Tukey Highest Significant Difference tests were applied to determine whether statistical significance (p < 0.05) was achieved for individual analytes between the controls and the three levels of ME/CFS severity, as defined by WST (Table 2 and Additional file 1: Table S11).

Table 2 Summary statistics for standing time (ST) and weighted standing time (WST) for the ME/CFS and healthy study control groups Full size table

Because of skewness in both standing time (ST) and weighted standing time (WST) data, Wilcoxon signed rank tests were employed to investigate significance for both median ST and median WST between groups (Table 2). The Wilcoxon signed rank test, Mann–Whitney U, one-way ANOVA and follow-up tests were conducted using R 3.3.0 [20].

Identification of diagnostic criteria that discriminate between categories of ME/CFS were done on the basis that no more than three of the healthy-mild patients recorded presence of the criterion.

Pathology and cytokine testing

All participant serum, blood and urine analyses were conducted by HealthScope Pathology (now Australian Clinical Laboratories), Clayton Victoria, Australia. Activin proteins and cytokines were measured in serum, as described previously [24,25,26].

Human ethics 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).