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Eur Child Adolesc Psychiatry (2018) 27:233–240 https://doi.org/10.1007/s00787-017-1039-2

ORIGINAL CONTRIBUTION

Hydrogen breath test to detect small intestinal bacterial overgrowth: a prevalence case–control study in autism

Li Wang1 · Yu‑Mei Yu1 · You‑qi Zhang1 · Jie Zhang1 · Na Lu1 · Na Liu2 

Received: 9 July 2016 / Accepted: 4 August 2017 / Published online: 10 August 2017 © Springer-Verlag GmbH Germany 2017

Keywords Small intestinal bacterial overgrowth · Autism spectrum disorders · Hydrogen glucose breath test · Chinese

Abbreviations ASD Autism spectrum disorder GI Gastrointestinal IBD Inflammatory bowel disease VTE Venous thromboembolism NBS Expanded newborn screening ADI-R Autism Diagnostic Interview-Revised SIBO Small intestine bacterial overgrowth (SIBO) ATEC Autism Treatment Evaluation Checklist BMI Body mass index NICU Neonatal intensive care unit 6-GSI 6-Gastrointestinal Severity Index ID Intellectual disability IQ Intelligence quotient H2 Hydrogen CH4 Methane SD Standard deviation IQR Interquartile ranges LPS Lipopolysaccharide

Introduction

The prevalence of autism spectrum disorders (ASDs) has increased over recent years in much of the Western world [1]. In China, relatively little is known about the prevalence of ASDs. The underlying causes remain unclear, but both genetic and environmental factors appear to play a role [2]. High proportions of children with autism suffer from gas- trointestinal (GI) symptoms, implying a link between autism and abnormalities in gut microbial functions [3]. A pilot

Abstract The aim of this study is to assess the prevalence of small intestinal bacterial overgrowth (SIBO) by hydro- gen breath test in patients with autism spectrum disorders (ASD) with respect to a consistent control group. From 2011 to 2013, 310 children with ASD and 1240 sex- and age-matched typical children were enrolled in this study to undergo glucose breath test. The study participants were considered to exhibit SIBO when an increase in H2 of ≥20 ppm or CH4 of ≥10 ppm with respect to the fast- ing value was observed up to 60  min after the ingestion of glucose. Ninety-six children with autism suffered from SIBO, giving a prevalence rate of SIBO was 31.0% (95% CI 25.8–36.1%). In contrast, 9.3% of the typical children acknowledged SIBO. The difference between groups was statistically significant (P < 0.0001). The median Autism Treatment Evaluation Checklist (ATEC) score in the chil- dren with autism and with SIBO was significantly high when compared with the children without autism and without SIBO [98 (IQR, 45–120) vs. 63 (32–94), P < 0.001]. For the autism group, the 6-GI Severity Index (6-GSI) score was found to be strongly and significantly correlated with the total ATEC score (r = 0.639, P < 0.0001). SIBO was significantly associated with worse symptoms of autism, demonstrating that children with SIBO may significantly contribute to symptoms of autism.

Li Wang and Yu-Mei Yu are co-first authors.

* Li Wang [email protected]

1 Department of Pediatrics, Cangzhou Central Hospital, No. 16, Xinhua West Road, Cangzhou 061000, Hebei, China

2 Department of Pediatrics, The Second Hospital of Hebei Medical University, Shijiazhuang, China

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study suggested the role of gut microbiota in autism as a part of the “gut-brain” axis [4]. Interestingly, de Theije et al. [5] showed that autism-like behavior and its intestinal pheno- type are associated with altered microbial colonization and activity in a murine model for ASD, with preponderance in male offspring. However, the data from another study did not support the hypothesis that the gastrointestinal microbiota of children with ASD plays a role in the symptomatology of ASD [6].

Small intestinal bacterial overgrowth (SIBO) occurs when colonic quantities of commensal bacteria are present in the small bowel. SIBO leads to impaired micronutrient absorption and increased GI permeability, both of which may contribute to celiac disease and stunting in children [7]. SIBO has been associated with irritable bowel syndrome [8], Parkinson’s disease [9], inflammatory bowel disease (IBD) [10], and venous thromboembolism (VTE) [11]. Interest- ingly, there is no study yet on the interaction between SIBO and children with ASD. The aim of this study was to assess the prevalence of SIBO by hydrogen breath test in patients with ASD with respect to a consistent control group.

Patients and methods

From 2011 to 2013, 310 children with ASD were enrolled in this study. Those children were all native and singleton live births who had taken part in expanded newborn screen- ing (NBS) in Beijing, China from 2008 to 2010, with out- patient follow-up when the children were 36 months old. All children will autism analysis. The Autism Diagnostic Interview-Revised (ADI-R) and DSM-5 criteria were used to confirm a diagnosis of ASD in outpatient follow-up. Those protocols were translated into Chinese Mandarin. Chinese- translated materials along with English materials were provided in advance. For each child with ASD diagnosis, we selected 4-to-1 sex- and age-matched controls in those typical children without ASD diagnosis. To exclude the pos- sibility that the controls could have any sub-clinical autistic features, each control subject was also clinically examined by the pediatrician. The Cangzhou Central Hospital Institu- tional Review Board for the Protection of Human Subjects approved this study. Neither data nor specimens were col- lected until written informed consents were obtained from the parents.

Sociodemographic factors [age, sex, body mass index (BMI), and ethnicity], infant characteristics [chronic com- plication before pregnancy, pregnancy-induced complica- tion, mode of delivery, and those transferred to neonatal intensive care unit (NICU) after delivery], family structure, area of residence (urban or rural), educational background of parents, NBS results, whether ASD diagnosis had been confirmed or expected before follow-up, and family history

of mental illness subdivided hierarchically as ASD were obtained from outpatient follow-up.

Autism severity was assessed with the Autism Treat- ment Evaluation Checklist (ATEC), which is an instrument designed to provide a quantitative assessment of autism severity [12]. ASD severity was divided into three groups, according to the ATEC score (mild <50; moderate 50–104; and severe 104–180). GI symptoms were assessed using a modified version of the GI Severity Index [13]. Specifically, we included only the first six items (constipation, diarrhea, stool consistency, stool smell, flatulence, and abdominal pain), but did not include “unexplained daytime irritabil- ity”, “nighttime awakening,” or “abdominal tenderness.” We call this shortened version the 6-Gastrointestinal Severity Index (6-GSI). Intellectual disability (ID) status was con- firmed using DSM-5 in the children in those processes [ID was defined as the IQ (Intelligence Quotient) <80]. IQ was assessed using the Combined Raven’s Test and then con- verted to a standard IQ score according to Chinese children’s norm.

Hydrogen and methane breath test

Glucose breath testing was performed under standard condi- tions. The glucose breath test was performed in the morn- ing following oral hygiene using 0.05% chlorhexidine. The children fasted for a period of 12 h prior to the test. Breath samples were collected using a non-rebreathing valve setup (QuinTron Instrument Co. Inc., Menomonee Falls, WI, US). After collection of the fasting breath, a dose of 50 g of glu- cose in the form of iso-osmotic solution was administered and samples were collected 15, 30, 45, 60, 90, 120, and 180 min after the ingestion of glucose. The levels of hydro- gen (H2) and methane (CH4) in the samples were simultane- ously measured by gas chromatography using a 12i model QuinTron MicroLyzer unit (QuinTron Instrument Company, Milwaukee, WI, US). The results were expressed in parts per million (ppm). Study participants were considered to exhibit SIBO when an increase in H2 of ≥20 ppm or CH4 of ≥10 ppm with respect to the fasting value was observed up to 60 min after the ingestion of glucose [14]. Determina- tions were performed in an independent laboratory blinded to clinical and sociodemographic data.

Data analysis

Results are expressed as percentages for categorical vari- ables and as means (standard deviation, SD) and medians (interquartile ranges, IQRs) for the continuous variables, depending on the normal or non-normal distribution of data. Shapiro–Wilk tests were used for normal distribution

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test. Proportions were compared using the Chi-square test. Two-group comparison of not normally distributed data was performed using the Mann–Whitney U test, and a two-tailed Student’s unpaired t test was used for normally distributed continuous variables.

Correlations among continuous variables were assessed by the Spearman rank-correlation coefficient. In addition, associations between the ATEC and 6-GSI scores were also assessed using logistic regression models in multivariate adjustment with possible confounders, such as sociode- mographic factors (age, sex, BMI, and ethnicity), infant characteristics (chronic complication before pregnancy, pregnancy-induced complication, mode of delivery, and those transferred to NICU after delivery), family structure, area of residence (urban or rural), educational background of parents, NBS results, whether ASD diagnosis had been confirmed or expected before follow-up, and family history of mental illness. All statistical analyses were performed with SPSS for Windows, version 20.0 (SPSS Inc., Chicago, IL, US). Statistical significance was defined as P < 0.05.

Results

In our study, 310 children with ASD were included. The male-to-female ratio was 3.43:1, and the ratios of autistic disorders to other ASD subtypes were 2.44:1. The rate of children who had ID was significantly lower in controls as compared with ASD groups (2.3 vs. 32.6%, P < 0.001).

Ninety-six children with autism suffered from SIBO, giv- ing a prevalence rate of 31.0% (95% CI 25.8–36.1%). In con- trast, 9.3% (115/1240; 95% CI 7.7–0.9%) of the typical chil- dren acknowledged SIBO (Table 1). The difference between groups was statistically significant (odds ratio 3.33; 95% CI 2.28–7.75; P < 0.0001). In the typical children group, we found that the rates of SIBO in rural and urban areas were

14.8 and 5.8%, respectively. In the group with autism, it was 50.5 and 21.9%, respectively.

The hydrogen concentrations (ppm) obtained by the glu- cose breath test were analyzed for the areas under the indi- vidual curves. The children with autism exhibited greater (Student’s t test, P < 0.001) mean hydrogen production dur- ing the first hour of the test, which presumably originated from the small intestine when compared with the typical children group (505.32 ± 315.46 vs. 302.14 ± 244.57 ppm per min, respectively). Between 60  min and 180  min of the test, the period during which hydrogen production occurs predominantly in the large intestine, the concentra- tion of hydrogen in the breath of the children in the ASD and control groups was similar (4576.77  ±  1012.55 vs. 4365.15 ± 997.52 ppm per min, respectively, P = 0.582) (Fig.  1a). Figure  1b shows the mean hydrogen concen- trations (ppm) obtained from the breath tests of children with and without bacterial overgrowth in the ASD group. A greater area under the curve for the small intestine was observed among the 96 children with SIBO compared with the 214 children without SIBO up to 60 min after the inges- tion of glucose (893.6 ± 505.56 ppm vs. 375.2 ± 177.1 ppm per min; Student’s t test, P < 0.001). A similar response was observed for the colon during the 60–180 min of the test (5128.9 ± 1262.5 vs. 4021.1 ± 711.8 ppm per min; Student’s t test, P < 0.001). Figure 2 shows the mean methane con- centrations (ppm) obtained from the breath tests of children with and without bacterial overgrowth in the ASD group. In the small intestine, differences in the area under the curve were not observed among the 96 children with SIBO in rela- tion to the 214 children without SIBO up to 60 min after the ingestion of glucose (P = 0.12). In addition, no significant difference was observed in the area under the curve for the colon during the 60 min to 180 min of the test (P = 0.08). Methane production was observed in 138 (64.5%) of the 214 children who did not exhibit bacterial overgrowth and

Table 1 The prevalence of SIBO in different groups

ASD autism spectrum disorders, ATEC Autism Treatment Evaluation Checklist, SIBO small intestinal bac- terial overgrowth  a ASD was divided into three group, according to ATEC score (mild <50; moderate 50–104; severe 104– 180). Proportions were compared using the Chi-square test  b P < 0.01 vs. typical children  c P < 0.05 vs. typical children

Cohort No. of children No. of with SIBO

Prevalence (%) 95% CI (%) OR (95% CI)

ASDa 310 96 31.0 25.8–36.1 3.33 (2.28–7.75)b

Mild 99 17 17.2 9.7–24.6 1.85 (1.29–3.05)c

Moderate 130 39 30.0 22.1–37.8 3.23 (2.10–6.93)b

Severe 81 40 49.4 38.5–62.3 5.31 (2.76–12.63)b

Typical children 1240 115 9.3 7.7–10.9 Reference

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in 60 (62.5%) of the 96 children with bacterial overgrowth (X2 test, P = 0.74).

Diarrhea was the most common SIBO symptom (71.0% of ASD patients), followed by abdominal pain (37.1%), and abnormal feces (30.0%). Children with autism and with SIBO were more likely from the rural area (50.0 vs. 21.9%; P  <  0.001), NBS positive (4.2 vs. 0.9%; P  =  0.046), ID (45.8 vs. 26.6%; P = 0.001), and transferred to NICU after delivery (16.7 vs. 2.4%; P < 0.001), Table 2. Table 3 shown that transferred to NICU, place of residence, NBS screening

result, ATEC score and 6-GSI were associated with hydro- gen concentrations (P < 0.05). Associations were not found between SIBO and sex, age, BMI, ethnicity, chronic compli- cation before pregnancy, pregnancy-induced complication, mode of delivery, family structure, educational background of parents, NBS results, whether ASD diagnosis had been confirmed or expected before follow-up, and family history of mental illness (P > 0.05; Table 2).

The median ATEC score in the children with autism was 75(IQR, 38-106). The median ATEC score in the children

Fig. 1 Mean concentrations of hydrogen (ppm) in breath samples in different groups. a Mean concentrations of hydro- gen (ppm) in breath samples collected after fasting and at 15, 30, 45, 60, 90, 150 and 180 min after glucose ingestion from children in the ASD (n = 310) and typical children (n = 1240) groups. Student’s t test, comparison between groups for collection time during the breath test (complementation of the analysis of area under the curve). aP < 0.05 vs. typical children group. b Mean concen- trations of hydrogen (ppm) in breath samples collected after fasting and at 15, 30, 60, 90, 120, 150 and 180 min after glu- cose ingestion from children in the autistic group with (n = 96) and without (n = 214) small intestinal bacterial overgrowth (SIBO). aP < 0.05 vs. the autistic group without SIBO in relation of the hydrogen produc- tion in breath test

Fig. 2 Mean concentrations of methane (ppm) in breath sam- ples collected after fasting and at 15, 30, 60, 90, 120, 150 and 180 min after glucose ingestion from children in the autistic group with (n = 96) and without (n = 214) SIBO. bP < 0.05 vs. the autistic group without SIBO in relation of the methane production in breath test

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with autism and with SIBO was significantly high when compared with the children with autism but without SIBO [98 (IQR, 45–120) vs. 63 (32–94), P < 0.001]. In addition, the prevalence of SIBO in the mild group was 17.2% (95%

CI 9.7–24.6%). The corresponding prevalence rates for the moderate and severe groups were 30.0 and 49.4%, respec- tively (Table 2). For the autism group, the median 6-GSI score was 4 (2–7), and the 6-GSI was found to be strongly and significantly correlated with the total ATEC score (r = 0.639, P < 0.0001). A significant positive correlation was also found between the 6-GSI and ATEC scores, using ordered logistic regression after multivariate adjustment for the abovementioned possible confounders (P < 0.001).

Discussion

Recent studies have correlated gut dysfunction with the ASD group and suggested a possible role of the GI microflora in the symptomatology and/or severity of symptoms in children with autism [15]. However, the evidence is rather specula- tive. To date, no study has directly examined the association between SIBO detected by hydrogen glucose breath test and ASD. We had the opportunity to examine this relationship in a large, population-based Chinese case–control study. We found that a higher proportion of ASD patients suffered from

Table 2 Baseline characteristics of the enrolled ASD with SIBO and without SIBOa

SIBO small intestinal bacterial overgrowth ASDs, autism spectrum disorders, NBS newborn screening, NICU neonatal intensive care unit, ID intellectual disability, ATEC Autism Treatment Evaluation Checklist, 6-GSI 6-GI Severity Index  a Two-group comparison was performed using the Chi-square test and Mann–Whitney U test or Student’s unpaired t test

SIBO(+) SIBO(−)

N 96 214 Han Chinese (%) 93.8 93.4 Preterm birth (<37 weeks),  (%) 11.5 11.2 Assisted delivery (%) 37.5 39.2 Parental depression (%) 12.5 10.8 Chronic complication before pregnancy (%) 10.4 10.2 Pregnancy-induced complication (%) 13.5 11.7 Transfer to NICU (%) 16.7 2.4 Sex of child, male (%) 81.3 75.7 Autistic disorders (%) 75.0 69.1 ID (%) 45.8 26.6 ASDs diagnosis had been confirmed or expected before follow-up (%) 27.1 22.9 NBS positive (%) 4.2 0.9 Live (rural, %) 50.0 21.9 Marital status, single (%) 9.4 8.4 Family’s socio-professional category, high (%) 11.5 9.8 Family history of ASDs (%) 8.3 9.9 ATEC score, IQR 98 (45–120) 63 (32–94) 6-GSI, IQR 6 (4–9) 2 (1–3) Hydrogen concentrations 60 min after glucose ingestion, mean (SD) 893.6 (505.5) 375.2 (177.1) Hydrogen concentrations 60–180 min after glucose ingestion, mean (SD) 5128.9 (1262.5) 4021.1 (711.8) Methane concentrations 60 min after glucose ingestion, mean (SD) 558.3 (332.2) 528.2 (324.1) Methane concentrations 60–180 min after glucose ingestion, mean (SD) 3045.2 (1102.3) 2905 (1054.5)

Table 3 The associations of hydrogen concentration with demo- graphic and health characteristics of the ASD group

NBS newborn screening, NICU neonatal intensive care unit, ATEC Autism Treatment Evaluation Checklist, 6-GSI 6-GI Severity Index

Characteristics r (Spearman) P

Race 0.08 >0.05 Mode of delivery 0.06 >0.05 Transferred to NICU 0.37 <0.001 Place of residence 0.28 <0.01 NBS screening result 0.32 <0.01 Marital status 0.09 >0.05 Family’s socio-professional

category 0.13 >0.05

Family history of ASDs 0.12 >0.05 ATEC score 0.48 <0.001 6-GSI 0.59 <0.001

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SIBO than in controls (31.0 vs. 9.3%, P < 0.001). A recent study of children with autism and their first-degree relatives found that 37 and 21%, respectively, had increased intestinal permeability based on a lactulose/mannitol test, compared to 5% of normal subjects [16]. Finegold et al. [17] used a high throughput sequencing technique, that is, pyrosequencing to investigate gut bacteria in children with autism vs. controls, and found several differences at the phylum level, includ- ing higher levels of Bacteroidetes in the group with severe autism, and higher levels of Firmicutes in the control group. Another study indicated that autistic subjects with gastroin- testinal disease harbor statistically significantly (P = 0.031) higher counts of C. perfringens in their gut compared to control children [18]. Furthermore, we found that SIBO was associated with worse symptoms of autism, demonstrating that children with more severe autism are more likely to suf- fer from SIBO. Similarly, Adams et al. [19] reported that the strong correlation of gastrointestinal symptoms with autism severity indicated that children with more severe autism are likely to have more severe gastrointestinal symptoms and vice versa. Therefore, it is possible that symptoms of autism are exacerbated or may even be partially due to the underly- ing gastrointestinal problems.

SIBO had been studied in different groups of children. Mello et al. [20] found that 21.6% of the children suffered from SIBO (those from slum areas vs. those from private schools: 30.9 vs. 2.4%). These values are similar to those previously reported in Brazil [21] for children living in slums and those attending private health clinics (37.5 and 2.1% SIBO in each group, respectively). In Australia, SIBO was found in 27.2% of aboriginal children fewer than 5 years old [22]. In our study, we found that the rates of SIBO in the rural and urban areas were 14.8 and 5.8%, respectively. Interestingly, we also found that 50.5% of the children with autism live in rural areas and suffered from SIBO, while 21.9% live in urban areas.

In a study of 137 children with ASD, 24% had a history of at least one gastrointestinal symptom, with diarrhea being the most prevalent one-occurring in 17% of individuals [23]. Similarly, a study of 172 children with autism spectrum dis- order found that 22.7% were positive for GI distress, primar- ily with diarrhea and constipation [24]. A characterization study of 160 children with ASD found that 59% had GI dysfunction with diarrhea or unformed stools, constipation, bloating, and/or gastroesophageal reflux (GERD)[25]. The exact percentage suffering from SIBO or GI problems varies from study to study, depending on the age of the study popu- lation and the different methodologies employed, but there is a general consensus that GI problems are common in autism.

The cause of these SIBO problems in autism is unclear, but it appears to partly relate to abnormal gut flora and possibly to the excessive use of oral antibiotics which can alter gut flora. Several studies have reported significantly

higher oral antibiotic use in children with autism vs. typi- cal children [19, 26]. In this study, we also found that children with autism and with SIBO were more frequently transferred to NICU after delivery (P = 0.028). In addition, Luna et al. [27] identified distinctive mucosal microbial signatures in ASD children with functional gastrointesti- nal disorders that correlate with cytokine and tryptophan homeostasis. However, Gondalia et al. [6] found no dif- ference between GI microbiota of children with autism and their neurotypical siblings. They suggested that other explanations for the gastrointestinal dysfunction in this population should be considered including elevated anxi- ety and self-restricted diets. More work should be carried out to assess the relationship between SIBO problems and autism.

Furthermore, we found that SIBO was associated with worse symptoms of autism. However, it is difficult to estab- lish whether the changes seen play a causative role or are merely a consequence of the disease. Interestingly, the effectiveness of oral, non-absorbable antibiotics in tem- porarily reducing symptoms of autism [28] suggests that the relationship may be causal, that is, we hypothesize that SIBO may significantly contribute to symptoms of autism in some children. Several possible mechanisms can be inferred. First, propionate has severe neurological effects in rats and Clostridia species are propionate producers. Studies by Mac- Fabe et al. [29] have demonstrated that injecting propionate directly into specific regions of rat brains in vivo can cause significant behavioral problems. Second, differences in the microbiota may also result in altered microbial metabolism of aromatic amino acids, with consequent changes in sys- temic metabolites (as reflected in urinary metabolite pro- files), which could lead to neurological symptoms [30]. Third, the microbiota could also be involved in the disease etiology via interactions with the immune system [31]. Some of the possible mechanisms outlined above are more likely to involve changes within the overall balance of the whole microbial community, while others may be exerted by spe- cific bacteria. Fourth, SIBO leads to steatorrhoea, vitamin B12 absorptive impairment and also injury to the small intestinal microvilli which itself causes malabsorption [32]. Zhang et al. [33] suggested that decrease in brain vitamin B12 status across the lifespan that may reflect an adaptation to increasing antioxidant demand, while accelerated deficits due to GSH deficiency may contribute to neurodevelopmen- tal and neuropsychiatric disorders. Finally, many pathogenic Gram-negative bacteria contain lipopolysaccharide (LPS) in their cell walls, which can cause damage in various tis- sues including the brain [3]. LPS-induced inflammation in the brain increases permeability of the blood–brain barrier and facilitates an accumulation of high levels of mercury in the cerebrum, which may aggravate ASD symptoms [34]. A test in rats showed that prenatal LPS exposure decreased

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levels of glutathione [35], which is an important antioxidant involved in heavy metal detoxification in the brain.

The strengths of our study include the fact that it is a prospective study with a relatively larger sample, making the results robust and generalizable. Furthermore, we col- lected data on a wide range of potentially confounding risk factors, allowing us to estimate the independent effect of SIBO on ASD. Finally, this is the first study which chose hydrogen glucose breath test to detect SIBO in children with ASD. This method is simple and efficient, and has a broad application prospects.

The following limitations of our study must also be considered. First, our sample is still relatively young, and the relationship between SIBO and later-onset ASDs will require revisiting in future years. Second, the results from even well-designed observational studies can be influenced by residual confounding. For example, socioeconomic status and dietary history would seem important, especially given that the ASD group with SIBO were more likely to require the NICU and show more severe symptoms of autism. How- ever, in this study, the information about socioeconomic sta- tus and dietary history were not obtained. Third, this was a prospective observational study; we could not determine whether SIBO treatment improves clinical manifestations in children with autism. Properly designed treatment tri- als are needed to confirm a causal link between SIBO and ASD. Interestingly, Kang et al. [36] suggested that alter the gut microbiome and virome can improve GI and behavio- ral symptoms of ASD. Furthermore, probiotics are hypoth- esized to positively impact gut microbial communities and alter the levels of specific potentially harmful metabolites in children with ASD [37]. Fourth, in addition, a decreased value of vitamin B12 is an important clinical problem of SIBO with potential neurological consequences [32, 33]. However, in this study we did not obtain the information about the dosage of vitamin B12 in the autistic children. Thus, we cannot determine the association of vitamin B12s with SIBO and autism. Finally, one of weakness of this study is in the assessment of SIBO itself, since there is no gold standard way to diagnose SIBO and the accuracy of all cur- rent tests, remains limited [38].

Conclusion

Children with ASD tend to suffer from severe SIBO prob- lems. SIBO was significantly associated with worse symp- toms of autism, demonstrating that children with SIBO may also significantly contribute to symptoms of autism. Strategies to treat SIBO or to improve gut microflora profile through dietary modulation may help to alleviate gut disor- ders common in children with autism.

Acknowledgements This work was supported by the National Natu- ral Science Foundation of China (30950031). The funding organiza- tions had no role in the design and concept of the study; the collection, management, analysis, and interpretation of the data; or the prepara- tion, review, or approval of the manuscript.

Compliance with ethical standards

Conflict of interest All authors have no conflicts of interest to dis- close.

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