Introduction

Autism spectrum disorders are neurodevelopmental disorders that comprise autism, Asperger’s syndrome, and pervasive developmental disorder not otherwise specified (PDD-NOS). It has many consequences in cognitive and sociability impairments, impairments, impairments in language and communication skills, restricted interests, and stereotyped behaviors (Johnson and Myers 2007).

A diagnosis of ASD is typically made before the age of 3 years, and the diagnoses have dramatically increased in the past years; ASD are about four times more prevalent in males than females, for unclear reasons (Centers for Disease Control and Prevention (CDC) 2014.

While the etiology of ASD remains unidentified, evolving evidence suggests multiple gene defects may be involved in tandem with an environmental catalyst (Cusco et al. 2009; Heberling et al. 2013). Gut microbiota have been incriminated because children with ASD often have gastrointestinal problems that correlate with ASD severity (Wang et al. 2011). Numerous previous studies have stated pathogenic gut bacteria in children with ASD (Li et al. 2017; Rosenfeld 2015; Wang et al. 2013).

Clostridium difficile may be present as a colonizing inhabitant of the normal gut microbiota of some individuals, however, provoke no visible signs of disease. Most cases of C. difficile infection appear in patients who are prescribed high-dose or long-course antibiotics, which disrupt the normal balance of the gut microbiota, changing its composition and leading to the overgrowth of C. difficile bacteria (Van den Abbeele et al. 2013).

The Clostridium hypothesis started by Sandler et al. (2000), in which children with regressive autism were treated with oral vancomycin for 6 weeks. Noteworthy improvement in GI and neurobehavioral symptoms was noticed in eight of the ten children, providing suggestion in favor of a toxin-producing Clostridium as a possible reason of regressive autism. However, gradual regression in bowel and behavioral symptoms occurred in all subjects following the discontinuation of vancomycin. This relapse was explained by the fact that many Clostridia can transform into spore-form, which are greatly resistant to antibiotics, but can then later germinate into vegetative, infective forms, and continue toxin production. Moreover, the fact that oral vancomycin’s effects are confined to the intestinal tract and not systemically absorbed provides further support for the involvement of the intestinal microbiota (Sandler et al. 2000).

Some studies endorse the Clostridium hypothesis, although the accurate species responsible have not been fully clarified (Finegold et al. 2002; Song et al. 2004).

In the present study; we aimed to assess Clostridium difficile in the stool of children with ASD and its relation to GI comorbidities, autism severity, and sensory impairment in our population.

Subjects and Methods

Fifty-eight autistic children, who presented to the Autism Clinic of Alexandria University Children’s Hospital, were enrolled in our study. These children were diagnosed with ASD stated by the diagnostic and statistical manual of mental disorders fifth edition (DSM-5) criteria (American Psychiatric Association (APA 2013). Their age ranged from 3 to 10 years. Forty- five neurotypical siblings and a cross- matching unrelated control group of 45 normally developing children of similar age and sex were also included.

Children with ASD with known syndromes, hepatic impairment, or immune deficiencies were excluded from the study.

Approval of the ethics committee of the Faculty of Medicine, Alexandria University, was obtained. Parents were explained about the study and written informed consent was obtained for their children’s examination and intervention.

All the studied children were subjected to thorough history taking and complete physical examination with special emphasis on neurological examination. The severity of autism was assessed by using Childhood Autism Rating Scale (CARS) (Rellini et al. 2004).

Presence or absence of sensory problems was assessed in children with ASD using short sensory profile (SSP) (Dunn 1999). The SSP is a 38-item caregiver report. Items were scored on a 1-point to a 5-point scale. The seven sections of the SSP were tactile sensitivity, taste/smell sensitivity, movement sensitivity, under responsive/ seek sensation, auditory filtering, low energy/weak, and visual/auditory sensitivity. Cases were classified as having definite sensory impairment, probable sensory impairment, or typical performance for each of these sections and a total score. Both section scores and a total score were interpreted on the SSP and were treated as the independent variables. The total score is the most sensitive indicator of sensory dysfunction.

Gastrointestinal (GI) symptoms were assessed using a modified short version of the GI Severity Index; 6-GSI questionnaire (Schneider et al. 2006). It consisted of six items (constipation, diarrhea, stool consistency, stool smell, flatulence, and abdominal pain). Every variant took a score of 0, 1, or 2 according to its frequency per week; zero scores were interpreted as the absence of the symptom while 1 and 2 denoted presence of the symptom with different severity. A total score equal or less than three was classified as low score and more than three was a high score.

C. difficile Detection

Specimen Collection, Preservation, and Transport

Stool specimens were collected, kept in the freezer upon defecation at home, and within the same day delivered to our laboratory frozen, where aliquots of each specimen were frozen at − 80 °C until DNA extraction in the same week.

DNA Extraction

DNA was extracted from 150-mg stool samples using ISOLATE Fecal DNA Kit (Bioline, UK) according to the manufacturers’ information. Fecal samples were added directly to a bashing beads lysis tube and they were rapidly lysed by bead beating in a vortex, without the use of organic denaturants or proteinases. The DNA was then bound, isolated, and purified using spin columns. The resulting DNA extracts were stored at − 80 °C until PCR assessment.

Detection and Quantitation of C. difficile

All the sequences of the primers were obtained from the previously published studies. Oligonucleotide primers targeted the 16S rRNA gene (rDNA) sequences of C. difficile were used (Penders et al. 2005). Primers were also used to amplify a conserved 16S rDNA sequence present in all bacteria (universal primer set, recognizing domain bacteria), and the amplification of which served as the denominator against which the amplification of the C. difficile was compared (Nadkarni et al. 2002). Primers were commercially obtained (Metabion International AG, Germany).

Amplification was performed in a light cycler (Rotor Gene Q, Qiagen, Germany) using a SensiFAST ™ SYBR No-ROX PCR kit (Bioline Co. UK). In brief, forward and reverse primers (4 pmol each) were used in 20 μl reactions containing 2 μl of the DNA extract.

PCR amplification was achieved with an initial denaturation at 95 °C for 10 min, followed by 40 cycles of denaturation for 30 s at 95 °C, annealing for 30 s at 60 °C, and extension for 30 s at 72 °C. Melting curve analysis was achieved from 40 to 95 °C with a plate reading step after every 1 °C and held at a particular temperature for 10 s to check the specificity of the product formed.

Quantitation of specific bacteria DNA is not expressed as an absolute number but expressed relative to total bacterial DNA present in the stool sample. Mean of relative abundance value of the bacteria is shown in instances in which the decimal value is low, as E−05 (4.75 × 10−5 is shown as 4.75E−05) (Balamurugan et al. 2008).

Detection of C. difficile Toxins A and B

This assay is based on the amplification of the genes encoding toxins A and B, the major virulence factors of this bacterial species. Amplification was accomplished by utilize 0.7 μM (each) primers tcdA442 and tcdA579, and 0.45 μM (each) primers tcdB2667 and tcdB2746 each in a separate reaction tube.

PCR amplification was performed with a primary denaturation at 95 °C for 3 min, followed by 45 cycles of denaturation at 95 °C for 10 s, annealing at 57 °C for 15 s, and extension at 72 °C for 7 s. Melting curve analysis was performed from 40 to 95 °C with a plate reading step after every 1 °C and held at a particular temperature for 10 s to check the specificity of the product formed (Belanger et al. 2003).

Statistical Analysis of the Data

Data were fed to the computer and analyzed using IBM SPSS software package version 20.0. (Armonk, NY: IBM Corp). Qualitative data were described using the number and percent. The Kolmogorov-Smirnov test was used to confirm the normality of distribution. Comparisons between groups for categorical variables were assessed using chi-square test (Fisher or Monte Carlo). The Student t test was used to compare two groups for normally distributed quantitative variables. Kruskal-Wallis test was used to compare different groups for abnormally distributed quantitative variables and the Mann-Whitney test was used to compare between two groups. Spearman coefficient was used to correlate between quantitative variables. Significance of the obtained results was judged at the 5% level.

Results

Out of the 58 ASD children, 39 (67.2%) were males and 19 (32.8%) were females with male to female ratio of 2.1:1. Their age ranged from 3 to 10 years with a mean of 5.41 ± 1.55 years. Out of the 45 siblings, 22 (48.9%) were males and 23 (51.1%) were females. The mean age was 4.31 ± 3.23 years and ranged from 0.5 to 12 years. For the unrelated control, out of 45 controls, 28 (62.2%) were males and 17 (37.8%) were females. The mean age was 5.36 ± 2.6 years and ranged from 2 to 12 years.

According to CARS, 46 (79.3%) of ASD patients were mild to moderate (CARS < 36), while the other 12 (20.7%) were severe ASD (CARS ≥ 36). The CARS range was 30–45 with mean 33.2.

Regarding the GI symptoms at the time of examination, all ASD cases had at least one GI symptom. The most frequent symptom was offensive stool odor (86.2%), and the least was diarrhea (12.1%). The mean of the Total GSI score was 3.95 ± 1.58; low in 19 (32.8%) cases and high in 39 (67.2%) cases. (Table 1).

Table 1 Distribution of children with ASD according to GSI score (n = 58)
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Table 2 demonstrates the SSP score in children with ASD. The highest definite abnormality detected in 91.4% of cases was in the under responsive/seeks sensation, while the lowest was in the visual/auditory sensitivity which was found in only 8.6% of children with ASD. The total score of SSP among the children with ASD showed definite impairment in 52 (89.7%) cases, probable impairment in 6 (10.3%) cases, and no one had the typical performance on the total score.

Table 2 Distribution of children with ASD according to SSP score (n = 58)
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Table 3 illustrates the correlation between CARS, 6-GSI, and SSP among children with ASD and shows that there was a significant positive correlation between CARS and 6-GSI. Also, there was a significant negative correlation between CARS and total score of SSP. However, there was no statistically significant correlation between 6-GSI and total score of SSP.

Table 3 Correlation between different parameters in autism group (n = 58)
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Clostridium difficile was detected in 15 out of 58 (25.9%) ASD cases, 18 (40%) and 7 (15.6%) out of 45 siblings and 45 unrelated control respectively. As regards the relative abundance of C. difficile in positive cases, the median was 6.98E−06, 4.19E−06, and 5.71E−06 among the ASD, siblings, and unrelated control respectively. Although the percentage of Clostridium difficile was higher in ASD cases and their siblings than the unrelated control, there was no statistically significant difference between the three groups either qualitative or quantitative results (Tables 4 and 5).

Table 4 Comparison between the studied groups according to qualitative results of C. difficile
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Table 5 Comparison between the studied groups according to quantitative results of C. difficile
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Regarding toxin production, C. difficile was classified as toxin A and/or B producers or non-toxigenic. Table 6 shows that 11 (73.3%), 14 (77.8%), and 5/7 (71.4%) of C. difficile in positive ASD, siblings, and unrelated control cases respectively are toxigenic. Among the positive ASD cases, there are no bacteria produce A and B toxins together; A−B+ producers are found in 4 (26.7%), A+B− were found in 7 (46.6%), and non-toxigenic (A−B−) were found in 4 (26.7%) patients. In siblings, there are 1 (5.6%) A+B+ producers, 4 (22.2%) A−B+ producers, 9 (50%) A+B− producers, and 4 (22.2%) non-toxigenic strains. In unrelated controls, there are no one produce A+B+, 2 (28.6%) A−B+ producers, 3 (42.9%) A+B− producers, and 2 (28.6%) non-toxigenic strains. Also, there was no statistically significant difference between the three groups (p = 0.232) (Table 6).

Table 6 Comparison between the studied groups according to C. difficile toxin production
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Studying the relation between the presence or absence of C. difficile in children with ASD with CARS and SSP and 6-GSI score revealed that there was no statistically significant difference between negative and positive cases for C. difficile and CARS or SSP or 6-GSI score (Table 7).

Table 7 Comparison between children with negative and positive C. difficile regarding CARS, SSP, and GI symptoms and GSI score in ASD group
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Discussion

Gut microbiota possess potential involvement in a range of neurodevelopmental disorders as well as its impact on behavior and mood (Diaz Heijtz et al. 2011). Oral vancomycin treatment has been also reported to temporarily reduce autism-associated behavioral abnormalities, although this study did not depend on microbiological detection of specific bacteria (Sandler et al. 2000).

In the present study using quantitative real-time PCR, 15 (25.9%) in ASD group were positive for C. difficile compared with 18 (40%) in siblings and 7 (15.6%) in the unrelated control group, although higher in ASD and their siblings, the results were statistically insignificant. Moreover, there was no statistically significant difference between the studied groups according to toxin A and/or B production. Similar findings were detected in other studies.

Finegold et al. (2010) investigated GI microbiota in 33 autistic children, 7 neurotypical siblings, and 8 unrelated controls by using pyrosequencing. They did not find Clostridia to be specifically prevalent in the stools of autistic individuals (Finegold et al. 2010). Similarly, Martirosian et al. (2011) in a study to assess the presence of C. difficile toxins, included 41 autistic children and 10 healthy children, isolated Clostridium spp. strains were subjected to sequencing in order to search for C. histolyticum and it showed negative results (Martirosian et al. 2011). Also, Gondalia et al. found that there was no significant difference in the bacterial composition of fecal material of the autistic group and their typically developing siblings (Gondalia et al. 2012).

In Egypt, the gut microbiome in autism is not well studied, and even C. difficile-associated diarrhea (CDAD) and asymptomatic carriage are still not well estimated especially in pediatric patients because the anaerobic of stool samples is not a routine in our country and to some extent is considered expensive.

Abd El-Wahab et al. (2016), in Egypt, conducted a study to determine the incidence of C. difficile as an etiology of antibiotic-associated diarrhea in 60 hospitalized children with a history of antibiotic intake. Stool samples were processed for C. difficile isolation and examined for C. difficile toxins A and B by the enzyme-linked immunosorbent assay. The reported CDAD in their hospital was 46.2%. The asymptomatic carriage rate with toxigenic C. difficile was 11.8%. No one of the control infants and children had diarrhea and all of them were negative for C. difficile organism and toxin which is much less than our results which may be attributed to using different methods for detection (Abd El-Wahab et al. 2016).

In contrast, Parracho et al., using fluorescence in situ hybridization (FISH) targeting Clostridium groups, reported that levels of the Clostridium histolyticum group of bacteria were higher in the ASD children compared with typical children. Also, there was no significant difference detected in this bacterial population (Clostridium clusters I and II) between the ASD children and their healthy siblings, but total Clostridium was higher in ASD group than sibling group (Parracho et al. 2005).

Luna et al. found a significant increase in several mucosa-associated Clostridiales detected in ASD children with functional gastrointestinal disorders. They took a rectal biopsy to detect Clostridiales (Luna et al. 2017).

This difference between the current study and previously mentioned studies could be explained by different techniques, different environment, small studied sample, or different samples.

In the current study, all cases had at least one GI symptom; the most frequent symptom was offensive stool odor and the least was diarrhea. In previous researches, the results are variable. The reported prevalence of GI symptoms in children with ASD has ranged from 9 to 70% or higher (Black et al. 2002; Horvath and Perman 2002).

A study done by Parracho et al. found a high proportion of ASD patients had GI disorders (91.4%). Diarrhea was the most common GI symptom (75.6%), followed by excess wind (55.2%), abdominal pain (46.6%), constipation (44.8%), and abnormal feces (43%). Some autistic individuals were noted to suffer from various GI problems, including both diarrhea and constipation (Parracho et al. 2005).

In another study by Wang et al., parents reported significantly more GI problems in children with ASD (42%). The two most common Gl problems in children with ASD were constipation (20%) and chronic diarrhea (19%). Increased autism symptom severity was associated with a higher score of GI problems (Wang et al. 2011). Moreover, Garrindo et al. found that functional constipation was the most frequent type of GI diseases in children with ASD (85%) (Gorrindo et al. 2012).

Wasilewska and Klukowski reported that the most common GI symptoms included overproduction of intestinal gasses/flatulence (60%), bloating (38%), abdominal pain (37.8%), diarrhea (28%), burping/belching (25%), gastroesophageal reflux symptoms (16%), and constipation (10%) (Wasilewska and Klukowski 2015).

In the present study, according to the 6-GSI in the ASD group, the total score was high (3.95), with 67.2% of cases had a high score and only 32.8% cases with a low score. There was no significant difference between positive and negative cases for C. difficile as regards the 6-GSI score. However, there was a positive correlation between CARS and GI symptoms.

This agrees with Adams et al. (2011), where the autism group was divided into two groups: low GI problems and high GI problems. The two groups were compared for gut bacteria and there were no significant differences. However, they stated that the strong correlation of the 6-GSI and the ATEC (Autism Treatment Evaluation Checklist) greatly demonstrates that gastrointestinal problems are associated with autism severity. The autism group with high GI problems had significantly higher scores on the ATEC versus the autism group with low GI problems (Adams et al. 2011).

In ASD children, the elevated rates of GI dysfunction might be due to influences other than GI microbiota. For example, it is well recognized that individuals with ASD have higher levels of distress and anxiety and stress activates the hypothalamic-pituitary-adrenal axis, resulting in the neuronal release of catecholamines; activating the sympathetic nervous system could affect the gut mucosa and the sympathetic efferents can impede gut motility proposing a mechanism for constipation (Drahota et al. 2011; Mazefsky et al. 2014).

According to the sensory evaluation in ASD group in the current study, the total sensory checklist score was 52(89.7%) with definite impairment and 6 (10.3%) with a probable difference, and no one met typical performance. The highest definite impairment included under responsiveness/seeks sensation (91.4%).

Similarly, sensory processing sections of the SSP done by Tomchek and Dunn (2007) reported clear differences in the ASD group in the underresponsive/seeks sensation (86.1%), auditory filtering (77.6%), tactile sensitivity (60.9%), and taste and smell sensitivity (54.1%). Other SSP sections had relatively lower percentages of described sensory processing differences in the definite difference range but still a greatly higher percentage than the typically developing group. Particularly, when probable and definite differences classifications were summed as an indicator of some degree of sensory processing differences, 95% of the sample of children with ASD was rated as having some degree of difference in sensory processing based on the SSP total score (Tomchek and Dunn 2007).

Leekam et al. reported that children with autism were more affected by sensory abnormalities than were children with language impairment and developmental disability. Two out of 33 children with autism (6%) were not affected by a sensory symptom, several symptoms in both the visual and in the auditory domains that appeared in 50% or more of the autism (Leekam et al. 2007).

In the current study, there was no relation between C. difficile and sensory impairment. However, there was a correlation between autism severity (CARS) and sensory impairment.

Gondalia et al. detected no significant differences within the ASD group when comparing those with and without GI dysfunction as regards the symptoms and the behavior (Gondalia et al. 2012). Moreover, Mazefsky et al. (2014) found that children with and without gastrointestinal problems did not vary in autism symptom severity, adaptive behavior scores, or mean social problem scores.

Conclusions

To summarize, Clostridium difficile is not specifically prevalent in the gut of children with ASD. Although most of the strains are toxigenic, there were no GI symptoms in the control groups and no statistically significant correlation with GI Severity Index in autistic cases. Gastrointestinal dysfunction and sensory impairment are common comorbidities in ASD.