Open-label pilot for treatment targeting gut dysbiosis in myalgic encephalomyelitis/chronic fatigue syndrome: neuropsychological symptoms and sex comparisons

ME/CFS is a complex, neuroimmune condition characterised by post-exertional mental and physical fatigue that is disproportionate to the level of exertion [1]. The multisystemic dysregulation results in pathophysiological abnormalities affecting a combination of central nervous, immune, gastrointestinal, energy metabolism, cardiovascular and respiratory systems manifesting in heterogeneous symptomatic presentations [1]. The history of diagnostic discrepancies (see [1‐4]) is reflected in varied prevalence rates between .08 and 2.6% [5‐11] but the burden on both the patient, their family and society is unequivocal [12]. This burden is not only a result of the devastating impact that the condition has on the patient’s daily, occupational and social functioning [13‐15] but can also be attributed to the direct cost of medical care that is often exacerbated by misdiagnosis and unclear treatment pathways [16, 17]. This awareness provides the rationale to examine the efficacy of treatments targeting pathophysiological abnormalities in ME/CFS patients with the hope of minimizing clinical exploration and identifying subgroups that may be more responsive to specific treatments.

Gastrointestinal disturbance and comorbid irritable bowel syndrome (IBS) are common in ME/CFS [18]. Estimates from a clinical sample of 1400 patients found that 80–90% experienced recurring gastrointestinal symptoms [19]. Intestinal permeability of the mucosal lining of the gastrointestinal tract [20, 21] and an imbalance in commensal enteric bacteria (i.e., gut dysbiosis) using culture-based methods (i.e., microbiota [22, 23]) and DNA sequencing (i.e., microbiome [24‐26]) have also been shown in this population. These imbalances in both the microbiota and microbiome appear distinct from healthy controls [24, 26], and associated with inflammation [25] and symptom expression [23, 26‐29]. Accumulating evidence suggests that microbial imbalances (whether consequential or causative) should not be viewed in isolation as they may be relevant for multiple ME/CFS symptoms, including but not limited to neurological manifestations.

Gut–brain interaction occurs through multiple bidirectional pathways including through central, autonomic, and enteric nervous systems; neuroendocrine and neuroimmune pathways; and enteric microbiota [30‐32]. Our understanding of the importance of the symbiotic relationship between enteric microbiota and health is becoming well accepted, with research efforts directed towards understanding mechanisms of microbial/host communication (see [33]). Gut dysbiosis may directly or indirectly precede gastrointestinal, neurocognitive and immune disturbances [34] or may be a consequence of stress and neurobiological mechanisms (e.g., in animal models [35‐37]). Results of antibiotic [27], probiotic [38‐40] and faecal transplant [41] interventions provide preliminary support for microbiota–gut–brain interactions in ME/CFS.

The d-lactate theory has been proposed as a possible mechanism for the neurological disturbances associated with gut dysbiosis in this population [23, 34, 42]. d-Lactic acidosis is an acute metabolic acidosis with associated encephalopathy that is observed in patients with a history of small bowel resections [43]. The shortened small bowel can lead to impaired absorption of carbohydrates, preferential growth of selected gut bacteria (e.g., increase in some species of Lactobacillus and Streptococcus) that promotes an acidic colonic environment and excess production of d-lactic acid [44]. This abundance of d-lactic acid combined with decreased metabolic capacity can lead to excess absorption within the blood and brain believed to play a role in the neurological symptoms of d-lactic acidosis [44]. Within ME/CFS, an overgrowth of Streptococcus and Enterococcus species (d-lactic acid producing bacteria) has been observed in culture-based microbial studies [23]. This bacterial imbalance, combined with overlapping neurological symptoms and possible mechanisms have contributed to the proposal that subclinical concentrations of d-lactate may play a role in ME/CFS presentations [42]. To date, measurement of d-lactate concentrations in ME/CFS have not been published.

In accordance with the d-lactate theory, an antibiotic treatment has been proposed to target the overgrowth of commensal enteric microbiota within the Streptococcus genus. Results from our group’s earlier pilot showed initial promise on some sleep and mood outcomes for a subgroup of participants who decreased in Streptococcus after 6 days of oral erythromycin treatment [27]. Other probiotic interventions used with ME/CFS patients may contradict the d-lactate hypothesis. Results indicating improved neurocognitive [38] and anxiety [39] symptoms using lactic acid-producing bacteria (predominantly Lactobacillus strains) question the mechanisms at play. Notably, colonic bacteria can produce d- and l-lactate with the ratio and rate of metabolism dependent on the species [45]. The proportion of d:l lactate produced by the bacterial strains used in the probiotic studies were not measured. The validity of the d-lactate theory as well as the efficacy of antibiotic and probiotic interventions in ME/CFS requires further examination.

Findings from our cross-sectional study correlating commensal microbiota and clinical symptoms in 274 ME/CFS patients [28] provided an interesting perspective on the role of d-lactate in males and females. Results showed small to moderate positive correlations for both Streptococcus and Lactobacillus with symptoms in males, suggesting that increased abundance of these genera were related to more impairment across several ME/CFS symptoms [28]. For Streptococcus, opposite associations were shown in females with small negative correlations suggesting that higher Streptococcus was associated with less pain, neurosensory and immune symptoms. These results highlighted the importance of considering sex differences in microbial function and supported the notion of the ‘microgenderome’, i.e. the critical role of sex hormones on host–microbiota interactions [46]. These findings also raised questions about the possibility of sex differences in response to oral erythromycin treatment targeting an overgrowth of Streptococcus.

This study is positioned within the context of d-lactate and microgenderome theories. In light of research yielding sex-specific correlations between Streptococci and ME/CFS symptoms [28], the objective of this study was to examine whether there was a sex-specific treatment response to an intervention designed to reduce the content of bacteria of the Streptococcus genus. Thus this pilot study compares the treatment response of male and female ME/CFS patients using a combined antibiotic and probiotic intervention aimed at reducing Streptococcus. Clinical outcomes measuring sleep, mood and cognitive symptoms were prioritised. The intervention was an extension of the earlier pilot [27] with alternate weeks of oral erythromycin and d-lactate-free probiotic supplementation across a 4-week period. Urine d-lactate and l-lactate concentrations were also measured to observe variation of lactate levels with this intervention to understand possible mechanisms of microbial–gut–brain interactions. To enable sufficient sample sizes for sex comparisons, an open-label design was used with the primary feasibility objective of determining the appropriateness of the intervention for both sexes rather than placebo control.

This open-label, non-randomised pilot used a repeated measures design with a baseline, intervention and post-intervention protocol across 6 weeks (see Table 1). The prospective intention was to recruit 40 patients with equal proportions of males and females to enable sex comparisons. Screening and recruitment was continuous, with consecutive commencement dates according to patient presentation at CFS Discovery Clinic, Melbourne, Australia.

New or current patients at the clinic aged above 18 years who met Canadian Consensus diagnostic Criteria for ME/CFS [47] were invited to be screened for participation in this study. Eligible participants were patients with Streptococcus viable counts above 3.00 × 10⁵ cfu/gm and more than 5% of the total count of aerobic microorganisms. Participants were asked to refrain from taking other antibiotics (from 4 weeks prior), probiotics (from 2 weeks prior), and substantially altering their diet, prescription medications or over-the-counter supplements across the screening and trial period. Known adverse reactions, contra-indications to the treatment protocol and/or significant comorbid physical or psychiatric illnesses excluded participation.

Trial methods were conducted in accordance with the guidelines for human experimental research and the Australian Clinical Trial Handbook [48]. Ethics approval was obtained from Victoria University Human Research Ethics Committee in June 2015 (HRE15-010). Additional trial details are available on the Australian and New Zealand Clinical Trial Registry (ACTRN12614001077651).

The treatment protocol combined antibiotic and probiotic therapy taken on alternate weeks. Tablets of Erythromycin 400 mg were given twice daily during weeks 2 and 4 (Erythromycin was given as the Ethyl Succinate salt and supplied by Amdipharm Mercury Pty Ltd or by Alphapharm Pty Ltd). Two capsules of Pro4-50 d-lactate free multistrain probiotic (Spectrumceuticals Pty Ltd, Belrose, New South Wales, Australia) were taken daily during weeks 3 and 5. Each probiotic capsule contained Lactobacillus rhamnosus (2.5 × 10¹⁰ cfu), Bifidobacterium lactis (1.5 × 10¹⁰ cfu), Bifidobacterium breve (5 × 10⁶ cfu), Bifidobacterium longum (5 × 10⁶ cfu). The off-label use of Erythromycin required notification to the Therapeutic Goods Administration under the Clinical Trial Notification scheme (Trial Number: 2015/0492) and approval was obtained on 29 June 2015.

Participants completed the intervention in their own homes. Compliance and adverse events were monitored with weekly phone calls throughout the intervention phase and participant completion of treatment adherence schedules.

Table 1 provides an overview of the timing of the outcomes assessed. Sleep patterns were measured objectively (actigraphy) using wrist Actiwatch monitors (Respironics Actiwear 2) that estimate movement and light. Participants completed a Response Booklet that included the sleep diary and self-report scales. Participants attended two external appointments for administration of the Cognitive Test Battery. The Cognitive Test Battery included measures of attention, memory, verbal fluency and executive functioning (see Additional file 1: Additional Method for additional details of all clinical measures and selected outcome variables).

The faecal microbial counts were performed on specimens that were preserved by cooling and then controlling the temperature until the commencement of laboratory analysis (see Additional file 1: Additional method). Classical cultural methods, on a variety of media, were used to perform the counts (see [28] for details of microbial identification and microbial quantification procedures). Identification of bacteria was performed by Matrix Assisted Laser Absorption and Ionisation Time of Flight Mass Spectrometry (MALDI-TOF–MS) using a proprietary peptide data base (MALDI Biotyper Bruker Daltonics, Bremen, Germany). Microbial variables included the count and relative abundance (RA) of selected aerobic (Streptococcus, Enterococcus, Escherichia) and anaerobic bacteria (Bacteroides, Bifidobacterium, Clostridium, Eubacterium, Lactobacillus). These variables were selected based on prior research [28]. RA_total was calculated by the ratio of each genus count divided by total detectable bacteria count (aerobic and anaerobic). The proportion of Streptococcus within total aerobic bacteria count (RA_aerobe) was also used as an outcome measure to be consistent with inclusion criteria and aid clinical interpretation.

The d-lactate and l-lactate concentrations in the urine samples were determined using High Performance Liquid Chromatography and Triple Quadrupole Mass Spectrometry (HPLC-TMS). Briefly, urine samples were acidified with hydrochloric acid and extracted with ethyl acetate. The ethyl acetate extracts were evaporated in a centrifugal vacuum evaporator. The residues were derivitised with an optically active reagent, (+)-O,O-diacetyl-l-tartaric anhydride, as originally described by Scheijen et al. [49]. These data are presented as the ratio of the concentrations of d-lactate to l-lactate. It is common to determine the ratio of analyte concentration in the urine sample the concentration of creatinine in the sample in order to correct for dilute or concentrated urine samples that arise from variation in the state of hydration of the subject. This was considered inappropriate in the current trial because there is evidence that the excretion of creatinine is increased in subjects suffering from ME/CFS (see [50]).

Primary and secondary endpoints were the change in scores on psychological outcomes at post-intervention for the intention-to-treat (ITT) population (i.e., all participants who commenced at baseline). A priori allocation of primary outcome status was based on evidence from research indicating sensitivity measuring treatment effects in this [27] and other clinical populations [51]. Primary outcome variables included a measure of sleep (actigraphic sleep efficiency; SE), mood (Profile of Mood States-Short Form Total Mood Disturbance, POMS [52]) and a measure of sustained visual attention (Rapid Visual Processing-A′, RVP-A′ from the Cambridge Neuropsychological Test Automated Battery, CANTAB [53]).

Multiple secondary endpoints were selected to evaluate change in microbiota (Streptococcus, Bifidobacteria and Lactobacillus count and RA), urinary d-lactate (d:l lactate ratio) and clinical symptoms including: objective sleep symptoms [Actigraphy sleep onset latency (SOL), wake after sleep onset (WASO), and restlessness/sleep fragmentation index (SFI)]; subjective sleep symptoms (Sleep Diary SOL, WASO, SE, and the Pittsburgh Sleep Quality Index, PSQI—Global Score [54]); mood (Depression, Anxiety and Stress Scale, DASS-21 [55]); cognition (word memory, story memory, spatial working memory, visual learning, verbal fluency, processing speed, cognitive flexibility and planning); fatigue (General Fatigue subscale from the Multidimensional Fatigue Inventory, MFI-20 [56]); and the Brain Fog subscale of the Multiple Fatigue Types Questionnaire, MTFQ [57]); and total symptoms (Symptom Severity and Symptom Hierarchy Profile, SSH-Total score [47]).

Uncertainty about the suitability of endpoints suggested a less hierarchical approach to outcome classification. Subsequently, the results of both primary and secondary outcome variables are presented together prioritising outcomes with large effect sizes (ES).

The study aimed to recruit equal proportions of males and females to conduct sex comparisons. Power analyses conducted by G*Power 3.1 indicated that the minimum sample size of n = 20 per group (alpha = .05, power = .8) would enable moderate to large ES estimates to achieve significance using analysis of variance (2 × 2 repeated measures ANOVA). A sample size of 40 for the combined group (alpha = .05, power = .8) was required to identify significant, moderate ES estimates using repeated measures ANOVA within factors (f = .23).

The results of primary analyses indicated the need for further investigation to understand outcomes and examine interactions between change in bacteria and change in symptoms. Correlations were chosen as the preferred method due to restrictions with sample size and violations of assumptions with other statistical techniques (i.e., MANOVA or regression). Proportional change scores were created for each clinical, microbial count and d:l lactate ratio variable using Eq. (1).

where X represents each variable analysed.

Therefore, scores of 100 reflect no change at post and numbers above or below reflect an increase or decrease at post, respectively. Spearman’s rho correlations (r_s) between change in clinical variables and change in microbial variables were chosen due to violations in normality. Missing cases were excluded pairwise. To allow for consistent interpretation of correlations, some r_s values were reversed (multiplied by − 1) so that a decrease in the clinical outcome score always represented improvement. Correlations were classified as small (.01), moderate (.03) and large (.05) effect sizes [66]. Only large effect sizes (i.e., r _s  > .05) were interpreted to reduce the risk of Type 1 errors from multiple correlations.

Figure 1 shows the participant flow diagram with 44 patients deemed eligible and consenting to participate from the 98 screened during recruitment (44.9%). A predominance of females (n = 27) were recruited compared with males (n = 17). The recruitment period was between 29th July 2015 and 8th November 2016. The date of the last data collection was 26th December 2016. All participants completed both baseline and post-intervention stages.

Baseline demographics for all participants are presented in Table 2. Participants were aged between 18 and 65 years with mean ages similar between the sexes. On average females spent less time working per week with 15/23 females (65.2%) not working compared with 5/14 males (35.7%). Higher mean scores of baseline general fatigue were reported by female (MFI-20, General Fatigue: M = 18.0, SD = 2.4) compared with male (MFI-20, General Fatigue: M = 15.7, SD = 3.4) participants although the range of scores on this subscale indicate individual variability across both groups. Mean scores for both sexes suggest ‘severe’ fatigue as indicated by scores above 13 [67]. (Possible scores on the MFI-20 range from 4 to 20 [56], and healthy controls average 6.8 on this subscale (see [67]). The mean years since diagnosis of ME/CFS was approximately 10 years for the total sample, female and male participants. The majority of participants (39/44) adhered to the treatment protocol (self-reported taking > 90% of the combined antibiotic and probiotic intervention).

Descriptive results, ES estimates and exact significance levels obtained from 2 × 2 ANOVAs are presented for the total ITT sample and stratified by sex (Additional file 1: Table S1). Some outcomes had missing data due to incomplete responses (questionnaires), collection error (stool and urine samples), and/or technical error (actigraphy). Management procedures for missing and ambiguous data are presented in the Additional file 1: Additional Method. Analysis of the change in scores from baseline to post for male and female subgroups (sex-time interactions) revealed no large effects and thus did not support a sex-specific response to the treatment (\(\mu_{p}^{2}\) < .014 for each outcome variable, see Additional file 1: Table S1). However, several dependent variables revealed a change across the intervention (i.e., time effects) when considering the sample as a whole. Figure 2 shows the ES estimates and confidence intervals for each outcome variable for the ITT sample.

The primary outcome for sleep, actigraphic sleep efficiency, revealed similar mean scores at baseline (M = 83.94, SD = 10.95) and post (M = 83.80, SD = 9.85) with a small effect estimate (\(\mu_{p}^{2}\) = .03, p < .297) indicating no change in objective measurement of sleep efficiency. However, small improvements in perceived (Diary) sleep efficiency (\(\mu_{p}^{2}\) = .14, p = .035) and sleep quality (PSQI: \(\mu_{p}^{2}\) = .15, p = .027) were shown. There was also a reduction in awakenings during the night with approximately 20% of the between-subjects variance accounted for by the intervention/time (actigraphy WASO: \(\mu_{p}^{2}\) = .21, p = .004; diary WASO: \(\mu_{p}^{2}\) = .20, p = .007).

Results for primary and secondary mood outcomes indicated minimal change in group mean scores across time. The primary mood outcome, POMS total score, revealed the lowest ES estimate (\(\mu_{p}^{2}\) = .01, p = .649) compared with DASS subscales (depression: \(\mu_{p}^{2}\) = .07, p = .009; anxiety: \(\mu_{p}^{2}\) = .04, p = .221; stress: \(\mu_{p}^{2}\) = .06, p = .151).

Five of the nine cognitive outcome variables revealed large ES estimates. The primary cognitive outcome, RVP A’, suggested an improvement in sustained attention from baseline (M = .91, SD = .42) to post (M = .94, SD = .04) with 53% of the between-subjects variance accounted for by the intervention/time (\(\mu_{p}^{2}\) = .53, p < .001). Secondary cognitive outcomes also indicated improvement across time in processing speed (\(\mu_{p}^{2}\) = .19, p = .004), cognitive flexibility (\(\mu_{p}^{2}\) = .43, p < .001), story memory (μ² = .21, p = .002), and verbal fluency (\(\mu_{p}^{2}\) = .14, p = .014).

The final clinical variable that suggested improvement was self-reported total symptoms (SSH) with group means reducing from baseline (M = 28.42, SD = 9.93) to post (M = 22.76, SD = 9.81) and approximately 29% of the between-subject variance attributed to the intervention/time (\(\mu_{p}^{2}\) = .29, p = .001). Notably, large sex effects were also observed for this variable with females (M = 31.14, SD = 8.16) reporting worse total symptoms compared to males (M = 23.00, SD = 11. 27) at baseline (\(\mu_{p}^{2}\) = .18, p = .015).

Streptococcus count was the only microbial variable that showed a large effect for time (\(\mu_{p}^{2}\) = .21, p = .003) with a reduction from baseline (M = 8.69 × 10⁶, SD = 6.39) to post (M = 6.88 × 10⁵, SD = 1.39 × 10²). No interaction, time or sex effects were observed on the d-lactate outcome variable. Interestingly split-plot graphs of Streptococcus count (Fig. 3a), RA_aerobe (Fig. 3b), and RA_total (Fig. 3c) showed a spread of individual responses to the treatment with several participants increasing at post (count = 12/42, RA_aerobe = 17/42, RA_total = 13/42). In addition to this individual variability, accurate interpretation of results from ITT analyses were limited by no placebo control and the possibility of practice effects on cognitive outcomes. To better understand associations between bacterial change and symptom expression the ancillary exploratory analyses were performed.

Results of non-parametric correlations of variable change scores for the total sample, males and females are presented in Additional file 1: Tables S3–S5. Detailed examination of the breadth of information provided by these ancillary analyses are beyond the scope of this paper. For the purposes of this article, only correlations with large effect sizes (r _s  > .5) are discussed to avoid over-interpretation with smaller samples and the risk of Type 1 error with multiple correlations. There were no large correlations between change in microbiota and clinical symptoms for the total sample (Additional file 1: Table S3). For females, results showed negative correlations (i.e., increased bacteria associated with clinical improvement) between change in: Clostridium and cognitive flexibility (r _s  = − .58, p = .002), Lactobacillus and planning (r _s  = −.50, p = .010), and Enterococcus and story memory (r _s  = −.50, p = .015; Additional file 1: Table S4). The majority of large correlations were shown for males (see Additional file 1: Table S5). Table 3 provides a summary of large correlations between change in clinical symptoms and microbial and lactate change variables in males.

The correlations presented in Table 3 indicate some consistency across several clinical outcomes for the genera Bacteroides, Bifidobacterium, Clostridium and d:l Lactate variables. Negative correlations suggest that an increase in Bacteroides (as observed in 11/16 males) was associated with improvements in sleep (Actigraphy WASO, Sleep Quality—PSQI), mood (Mood Disturbance—POMS Total; Stress—DASS), general fatigue (MFI-GF) and total symptoms (SSH). An association in the opposite direction was found for change on the cognitive measure of planning (SWM-Strategy), which was reduced.

Negative correlations were shown between change in Bifidobacterium and sleep quality (PSQI), general fatigue (MFI), anxiety (DASS), and visual learning. Alternatively, positive correlations were revealed between change in Clostridium and total symptoms (SSH) and some cognitive outcomes (verbal fluency, story memory, processing speed). Notably, change in Streptococcus correlated negatively with perceived sleep onset (Diary SOL) indicating that reduced Streptococcus was associated with subjectively longer time taken to fall asleep in males.

A small, negative correlation was observed between change in d:l lactate concentration ratios and change in Streptococcus count for the total sample (r _s  = − .243, p = .142). Correlations with clinical symptoms revealed that the change in d:l lactate concentration ratios was positively associated with change in sleep onset latency (actigraphy SOL), mood disturbance (POMS total) general fatigue (MFI) and total symptoms (SSH) in males. This would suggest proportionally higher concentrations of d-lactate were associated with adverse symptoms in males. Proportionally higher concentrations of d-lactate were seen in 9/15 males and 12/23 females at post intervention.

Six unexpected adverse events were reported from five participants. One participant (a) experienced severe diarrhoea, vomiting and cramping after taking the first antibiotic. This participant also experienced a respiratory allergic reaction to a non-protocol medication taken to attempt to relieve the gastrointestinal symptoms. Four other participants experienced an adverse event including (b) blood in stool (bloating but no pain reported), (c) difficulty sleeping, (d) rash on torso, and (e) exacerbation of Seborrheic dermatitis. Of these participants, the first (a) discontinued all treatment after the first antibiotic dose. The other participants (b) completed the treatment protocol, (c) reduced antibiotics (consumed 20/24 capsules), or reduced probiotics (d: consumed 11/28 capsules, e: consumed 14/28 capsules), respectively. All participants participated in post-intervention assessments.

The results of ITT outcome and ancillary analyses showing no change in d:l lactate ratio at post and small negative correlations between change in d:l lactate and Streptococcus, raise doubts about d-lactate metabolism from Streptococcal species. Considering, 21/38 participants increased in d:l lactate ratio after the intervention, it appears that the reduction of Streptococcus did not decrease d-lactate concentrations as expected. Given the enteric microbiota consists of more than 1000 species of bacteria [33], the limitations with culture-based identification methods, and the uncertainty around which species are producing lactate, it is possible that a reduction in Streptococcus may have allowed another d-lactate producing organism to proliferate. Some ancillary results provide partial support for d-lactate theory in males with change scores indicating decrease of d:l lactate ratio associated with improvement on some clinical outcomes (sleep onset (actigraphy SOL), mood disturbance (POMS), general fatigue (MFI), and total symptoms (SSH)). Perhaps our results reflect the relative change in reduced l-lactate production that would impact the ratio measured. Further research is needed to compare d-lactate concentrations (optimally in urine, faecal and serum samples) in ME/CFS with healthy controls and investigate other possible d-lactate producing bacteria, to adequately evaluate the relevance of the d-lactate hypothesis for either sex.

Our interpretation of d:l lactate is restricted by methodological limitations requiring the use of a lactate ratio. The routine use of creatinine for normalising urinary metabolites [72] may be inappropriate considering findings of higher creatinine concentrations in ME/CFS patients compared with controls [50]. Without an appropriate method for normalisation, absolute d-lactate concentrations and absolute l-lactate concentrations could not be statistically analysed because of the known wide variation in the concentration of spot urine samples in contrast to 24 h timed collections used to calculate daily excretion rates. Similarly, using genera rather than species data for microbial outcomes has reduced specificity and restricts interpretation.

The open-label design without placebo-control and using repeated measures carries inherent limitations restricting interpretation and generalisability of findings. Whilst the placebo response appears to be lower in ME/CFS than other medical conditions (e.g., depression, migraine, gastro-intestinal conditions), the influence of participant expectation appears to be greater for interventions with physiological targets (i.e., infectious or immunological) compared with psychosocial interventions in ME/CFS [73]. Discrepancies between cognitive measures and other symptoms raise questions about the influence of practice effects inherent in repeated testing over a short interval. Whilst alternate forms and outcomes with reduced practice effects were prioritised (see Additional file 1: Additional Method), ideally, controlled comparison can be used in future research to ascertain the proportion of change that can be attributed to familiarity with cognitive tests.

Other confounding factors included the influence of diet, concurrent medication and fluctuating symptomatology. Whilst we attempted to control for these factors by asking participants to remain stable on their diet and medication, the possibility of effects from other treatments or dietary intake cannot be excluded. The nature of the condition is that it has symptomatology that can be exacerbated or diminished without clear attributional cause. These fluctuations and other environmental (change in education or employment status, family stressors) and/or physiological (e.g., stage of menstrual cycle, viral/bacterial exposure) factors could not be controlled.

Statistical limitations include reduced power with smaller male samples, consideration of multiplicity of analyses and restricted interpretation with correlations. Results from correlational data only provide information about monotonic relationships, cannot attribute causation and have limited capacity to infer direct treatment effects. Cautious interpretations have been made focusing on large effects to attempt to reduce bias and improve generalisability. However, this conservative approach excludes small and moderate correlations that may also be relevant.

Some lactate results that contradict d-lactate theory prompt consideration of whether Streptococcus spp. or the intervention could have other modes of action. Streptococcal throat infections have been proposed as precipitating encephalitis and neurological symptoms in childhood (see [74‐76]). Evidence of abnormal basal ganglia imaging and antibasal ganglia antibodies suggests that streptococcal infections may trigger autoimmune responses in some individuals [76]. Within the context of ME/CFS, it seems reasonable to explore whether the overgrowth of commensal enteric Streptococcus, as observed in 58/92 (59.2%) patients screened, may exert immunological or autoimmune effects that contribute to neurological symptoms. Future research could also evaluate a history of streptococci infections and monitor immune and inflammatory markers to establish whether similar mechanisms are at play in ME/CFS. Monitoring immune and inflammatory markers could be particularly beneficial considering antibiotic macrolides have immune-modulating properties that may be a mechanism responsible for improvement in this clinical sample (see [77]).

Another possible mechanism of the intervention is through the prokinetic qualities of erythromycin. Erythromycin is a macrolide that inhibits protein synthesis in specific bacteria [78] and can increase gastric motility [79]. Low doses of erythromycin have been used for its prokinetic qualities in patients with delayed gastric emptying [80]. The stimulation of oesophageal, gastric and small intestinal contractions are likely to partially explain commonly reported gastrointestinal side effects (i.e., diarrhoea, nausea, vomiting) of oral erythromycin (see [81]). Therefore, the prokinetic effect of erythromycin may be particularly beneficial for this sample when we consider that constipation is a common symptom for patients with comorbid IBS and/or small intestinal bacterial overgrowth (SIBO; [82]), and the prevalence of intestinal permeability in ME/CFS [20, 21]. Increased monitoring of gastrointestinal changes, SIBO and IBS symptoms would be useful in further studies.

Probiotics may also increase bowel transit [83] or have other modes of action. Possible mechanisms of probiotics include modulating inflammatory and immune responses through enhancing the epithelial barrier, adherence to the mucosal wall, direct (antimicrobial) or indirect (competitive exclusion) effects on pathogenic microbiota, and vagal signalling (see [33, 84‐86]). Metabolic by-products from specific bacterial strains may also effect clinical presentations through the production of neurotransmitters (see [87]), short chain fatty acids through fermentation (see [33]), and cross-feeding, as discussed above. Advances in metabolomics methods would be useful to monitor functional changes during probiotic supplementation in ME/CFS patients.