Improvements in GI and ASD symptoms remained two years after the MTT stopped

Two years after the MTT was completed, we invited the 18 original subjects in our treatment group to participate in a follow-up study, and all provided informed consent. We performed the same GI and behavior tests that we employed previously19. 12 of 18 participants made some changes to their medication, diet, or nutritional supplements, but these changes were well documented and were mostly minor (Supplementary Table S1). We note that due to the open-label nature of this initial trial, all of the assessments are subject to placebo effect, however the long-term improvements we observed here are promising. Two years after treatment, most participants reported GI symptoms remaining improved compared to baseline (Fig. 1a and Supplementary Fig. S1). The improvement was on average 58% reduction in Gastrointestinal Symptom Rating Scale (GSRS) and 26% reduction in % days of abnormal stools (Daily Stool Record or DSR) relative to baseline, and this result is similar to what we observed at the end of treatment. The improvement in GI symptoms was observed for all sub-categories of GSRS (abdominal pain, indigestion, diarrhea, and constipation, Supplementary Fig. S2a) as well as for all sub-categories of DSR (no stool, hard stool, and soft/liquid stool, Supplementary Fig. S2b), although the degree of improvement on indigestion symptom (a sub-category of GSRS) was reduced after 2 years compared with weeks 10 and 18. This achievement is notable, because all 18 participants reported that they had had chronic GI problems (chronic constipation and/or diarrhea) since infancy, without any period of normal GI health (Supplementary Table S2). The families generally reported that ASD-related symptoms had slowly, steadily improved since week 18 of the Phase 1 trial, and this was consistent with the data reported in Fig. 1b–f. Based on the Childhood Autism Rating Scale (CARS) rated by a professional evaluator, the severity of ASD at the two-year follow-up was 47% lower than baseline (Fig. 1b), compared to 23% lower at the end of week 10. At the beginning of the open-label trial, 83% of participants rated in the severe ASD diagnosis per the CARS (Fig. 2a). At the two-year follow-up, only 17% were rated as severe, 39% were in the mild to moderate range, and 44% of participants were below the ASD diagnostic cut-off scores (Fig. 2a). The parent-rated Social Responsiveness Scale (SRS) assessment revealed that 89% of participants were in the severe range at the beginning of the trial, but the percentile dropped to 47% at the two-year follow-up (Fig. 2b), with 35% in the mild/moderate range and 18% below the cut-off for ASD. For the parent-rated Aberrant Behavior Checklist (ABC), total scores continued to improve, and were 35% lower relative to baseline (versus 24% lower at the end of treatment, relative to baseline; Fig. 1d). The Parent Global Impressions-III (PGI-III) scores remained similar to the scores at the end of treatment (week 10) of the open-label (Fig. 1e). The Vineland Adaptive Behavior Scale (VABS) equivalent age continued to improve (Fig. 1f), although not as quickly as during the treatment, resulting in an increase of 2.5 years over 2 years, which is much faster than typical for the ASD population, whose developmental age was only 49% of their physical age at the start of this study. Moreover, we observed improvement in behaviors in most sub-categories (Supplementary Figs S2c,d, and S3 for ABC, SRS, and VABS, respectively).

Figure 1 Changes in GI- and ASD-related symptoms of 18 children with ASD at two-year follow-up after treatment stopped. Asterisks (at the top of the box plot) indicate whether individuals (at each time point) have significantly changed since pre-treatment (Week 0 of original Phase 1 trial). Based on two-tailed Wilcoxon signed-rank test, ns indicates not significant, single asterisk indicates p < 0.05, double asterisks indicate p < 0.01, triple asterisks indicate p < 0.001. See also Supplementary Figs S1–S3. Full size image

Figure 2 CARS and SRS diagnostic category for ASD at baseline, 8 weeks after treatment, and two-year follow-up after treatment stopped. (a) For CARS, Minimal-to-No Symptoms (15–29.5 for ages less than 13; 15–27.5 for ages 13 or older), Mild-to-Moderate Symptoms (30–36.5 for ages less than 13; 28–34.5 for ages 13 or order), and Sever Symptoms (37 and higher for ages less than 13; 35 and higher for ages 13 or order)54. (b) For SRS, Normal (0–53), Mild or High Functioning autism (54–86), Clinical diagnosis of autistic disorder, Asperger’s disorder, or more severe cases of Pervasive developmental disorder not otherwise specified (PDD-NOS) (>87)55. (c–g) Strong and significant correlations between improvements in GI symptoms (GSRS) and behavior symptoms based on % changes in 2 years. Full size image

Overall, the most substantial improvements observed were on the CARS assessments, which was conducted by a professional evaluator and is less susceptible to placebo-effect20. CARS is a stable and consistent diagnostic tool with high predictive validity21 and has been used to evaluate participants before and after therapeutic interventions in multiple studies20,22,23. For the follow up CARS, the evaluator collected current information based on each question’s unique criteria. After the interview was complete for each question, the evaluator reviewed the information initially collected at baseline and used it for calibrating the final evaluation.

Improvements in GI and ASD symptoms were significantly correlated

We performed statistical analyses to assess whether improvements in GI and ASD severity were correlated. As shown in Fig. 2c–e, percentage changes in CARS, SRS, and ABC scores were positively correlated with percent changes in GSRS scores (Spearman correlation test, 2-tailed p < 0.005 and r > 0.7), implying that GI relief provided by MTT may ameliorate behavioral severity in children with ASD, or vice versa, or that both may be similarly impacted by another factor. Another GI assessment, DSR, however, showed that there was no significant correlation. Although the direction of the influence is not clear, a potential clinical link between GI and behavior severity is consistent with what previous studies have reported24,25.

ASD fecal bacterial diversity was higher two years after the MTT stopped

16 out of 18 original ASD participants provided an additional fecal sample two years after the open-label trial. Based on 16S ribosomal RNA (rRNA) gene amplicon sequencing analysis, most participants maintained higher gut microbiota diversity two years after treatment relative to baseline. Interestingly, for many individuals, the bacterial diversity was higher at two years than at the week 18 follow up as measured by Faith’s Phylogenetic Diversity (Fig. 3a and Supplementary Fig. S4a) and Observed OTUs (Supplementary Fig. S5a). Considering low gut bacterial diversity in individuals with ASD26 and other human disorders27,28,29, an increase in diversity after MTT may reflect that MTT intervention successfully transformed gut environment into a healthier status and led to a long-term benefit on GI and behavior symptoms.

Figure 3 Stool microbiota assessments at two-year follow-up after treatment stopped. (a) Faith’s phylogenetic diversity (PD) in the microbiota of 18 children with ASD as measured from stool samples. Orange lines indicate median PD of the donor samples (dashed line represents initial donor samples (n = 5), and dotted line represents maintenance dose samples (n = 2)), and green line indicates median PD of 20 neurotypical controls at week 0. ns indicates not significant, single asterisk indicates p < 0.05, double asterisks indicate p < 0.01, triple asterisks indicate p < 0.001 (two-tailed Wilcoxon signed-rank test comparing weeks 3, 10, and 18 and two-year to week 0 values). (b) Unweighted UniFrac distances between ASD gut microbiota and most relevant donor sample (initial donor sample at weeks 0 and 3, most recent maintenance dose sample at weeks 10 and 18, and 2 years). Green line indicates the median interpersonal variation between neurotypical controls and illustrates that prior to treatment the difference in gut microbiota composition between MTT recipients and donors was on the order of normal interpersonal variation. Statistics are the same as those used in (a). See also Supplementary Figs S5 and S6. Full size image

Upon completion of the original MTT treatment, we observed that the unweighted UniFrac distance30 between the gut microbiota of MTT recipients and their corresponding donors was smaller than before treatment, suggesting some engraftment of the donor microbiome into the recipients by MTT19. Interestingly, two years after the trial, the recipients were as different from the donor microbiome as they were pre-treatment as measured by unweighted UniFrac distance (Fig. 3b, Supplementary Fig. S4b) and several other metrics of community dissimilarity (Supplementary Fig. S5b–e). This suggests that the recipients didn’t retain completely the donated microbiome, but rather retained some features of it such as increased overall diversity, and increase in some important microbes such as Prevotella, while finding a new state.

Bifidobacterium and Prevotella relative abundances remained higher in feces of participants with ASD two-years after MTT stopped

Three taxa that were noticeably enhanced in MTT recipients at the conclusion of the original clinical trial19 were revisited during the two-year follow-up. Notably, compared to baseline, median relative abundances of Bifidobacteria and Prevotella increased 4-fold and 712-fold at week 10, and 5-fold and 84-fold at two years, respectively (Fig. 4a,b). Desulfobivrio relative abundance decreased since week 18 (Fig. 4c), but at the two-year follow-up was still marginally higher compared to baseline (two-tailed Wilcoxon signed-rank test, p = 0.07) and higher than neurotypical controls (two-tailed Mann-Whitney U test, p < 0.05). An increase in Prevotella after MTT is noteworthy, since its lower abundance in feces of children with ASD compared with neurotypical children has been confirmed in two different cohorts26,31. A recent study also found reduced levels of Prevotella in the oral microbiome of children with ASD32. Prevotella may be involved in butyrate production33, a key nutrient for the intestinal epithelial cells34. In addition, its co-occurrence with Desulfovibrio may reflect a synergistic advantage to outcompete other commensal microbes that utilize mucin as nutrients35, although more research is needed on how their ecological niche in mucin desulfation could contribute to an integrity of gut epithelial cells36 as well as to the improvement on GI and behavior symptoms we observed. Further mechanistic studies with multi-omic approaches are warranted to define the roles of Prevotella and Desulfovibrio in the context of autism.

Figure 4 Changes in relative abundances of Bifidobacterium, Prevotella, and Desulfovibrio. ns indicates not significant, single asterisk indicates p < 0.05 and double asterisks indicate p < 0.01 (two-tailed Wilcoxon signed-rank test comparing weeks 3, 10, and 8 and two-year to week 0 values). Orange lines indicate median of the donor samples (dashed line represents initial donor samples, and dotted line represents maintenance dose samples), and green line indicates median of 20 neurotypical controls at week 0. Full size image

Further research with a placebo-control arm is warranted

To the best of our knowledge, long-term follow-up studies are rare for medical treatment of individuals with ASD. In treatments with vancomycin37 or phytochemical sulforaphane38, benefits were lost within two or four weeks, respectively, of the treatments being discontinued. Thus, the long-term benefits observed here two years after MTT stopped are very encouraging, and MTT-driven gut microbiota transformation seems robust and long-lasting for the treatment of ASD. Despite steady and continuous improvement in behaviors over two years, we must underscore that the original clinical trial and current follow-up study are open-label trials without a control for placebo effect. Autism symptoms are relatively stable over time without a major intervention: for example, a trajectory study with 345 children with ASD showed that more than 80% of participants with ASD retained unexpectedly stable core symptoms severity over 8 to 12 years39. The VABS observations indicate that the improvements in adaptive behaviors observed here were substantially more than expected for children with ASD over two years. A limitation of this study is that 12 out of 18 participants made one or more changes to their medications, nutritional supplements, and diets between the end of the original MTT trial and the two-year follow-up since the treatment stopped (Supplementary Table S1). As described in detail in the methods section, participants were asked to rate the perceived effectiveness on GI and ASD symptoms (on a scale of 0–4) caused by changes in medications, diet, or nutritional supplements. Although the scale on the perceived effectiveness is still subjective and difficult to interpret, low scores received (1.1 for GI and 0.8 for ASD symptoms) suggest that these treatments on average could have only “slight effect”. Thus, it appears that most of the changes observed were probably due to the MTT, although we still need follow-up studies to understand whether the improvement by MTT were solely from vancomycin, MoviPrep, Prilosec, Standardized Human Gut Microbiota (SHGM), or a combination of these individual factors. For example, some participants in our study could have GI symptoms that were acid-peptic in nature, and their improvements on GI symptoms might be solely attributed to the administration of stomach-acid suppressant (Prilosec)40. We hypothesize that MTT may also be beneficial for children with ASD who do not have obvious GI symptoms but have low diversity of gut bacteria, as our previous study26 found that most children with ASD had low gut bacterial diversity, regardless of whether they have GI problems.

Here, we also would like to address a potential study limitation interpreting the improvement on GI symptoms after MTT, since these heterogeneous GI symptoms could reflect a wide range of underlying etiological GI pathologies. Although we reviewed medical histories to exclude children with known gastrointestinal diagnoses (such as ulcerative colitis, Crohn’s Disease, celiac disease, eosinophilic gastritis, or similar conditions)19, we did not conduct additional GI diagnostic evaluations, which is a limitation of this study. Thus, we want to underscore need of follow-up studies embracing more thorough examination of participants’ GI pathologies in order to better understand effectiveness of MTT.