Analysis of gut microbiome, nutrition and immune status in autism spectrum disorder: a case-control study in Ecuador

Figures & data

Table 1. Socioeconomic characteristics and early life history of children with autism and neurotypical controls.

	ASD (%)	Control (%)	p-value
ADI-R SCQ under 15	25(100.0) 0.0(0.0)	0.0(0.0) 35(100.0)
Male (%) Female (%)	24(96) 1(4.0)	32(91.4) 3(8.6)
Ethnic group Mestizo	25(100)	35(100)
Mean age (years)±SD	8.92 ± 2.3	8.46 ± 2.6	NS
Parents education level (Father/Mother)
Elementary School	2(8.0)/2(8.0)	4(11.5)/1(2.9)	NS
High school	4(16.0)/6(24.0)	6(17.1)/9(25.7)
University	18(72)/17(68)	25(71)/25(71)
Family income
$1500 or more	1(4.0)	15(42.9)	0.038
$700 to $1499	18(72.0)	15(42.9)
$200 to $600	6(24.0)	5(14)
Antibiotics during pregnancy
Yes	5(20.0)	2(5.7)	0.099
No	20(80.0)	33(94.3)
Route of delivery
Cesarean section	17(68.0)	13(37.1)	0.04
Vaginal	8(32.0)	22(62.9)
Breastfeeding > 4 months
Yes	20(80.0)	29(82.9)	NS
No	5(20.0)	6(17.1)
Formula-feeding
Yes	18(72.0)	22(62.9)	NS
No	7(28.0)	13(37.1)

ASD Autism spectrum disorder; ADI-R Autism Diagnostics Interview – Revised; SCQ Social Communication Questionnaire; SD standard deviation; NS non-significant.

Table 2. Comparison of BMI and blood biochemical parameters between children with autism and neurotypical controls.

Variable	ASD n = 25 (%)	Control n = 34 (%)	p-value
BMI for age
<-2 z-score – Underweight	2(8)	0(0)	0.103
≥-2/≤1 z-score – Normal	19(76)	32(94.1)	0.012
>1/≤2 z-score – Overweight	3(12)	2(5.9)	0.330
>2 z-score – Obesity	1(4)	0(0)	0.263
Glucose and lipid profiles mean±SD
Glucose (mg/dl)	88.3 ± 6.2	82.7 ± 21.7	0.228
Cholesterol (mg/dl)	156 ± 29.6	159.9 ± 31.7	0.645
HDL (mg/dl)	49.3 ± 11.7	56.3 ± 14.4	0.055
VLDL (mg/dl)	17.7 ± 10.9	13.7 ± 5.8	0.087
LDL (mg/dl)	89.1 ± 26.7	90.7 ± 26.5	0.827
Triglycerides (mg/dl)	88.6 ± 54.9	68.8 ± 28.9	0.083

ASD Autism spectrum disorder; BMI body mass index; HDL high-density lipoprotein; LDL low-density lipoprotein; VLDL very low-density lipoprotein; SD standard deviation.

Data expressed as mean±SD. Data were analyzed using analysis of variance – ANOVA.

Table 3. Mean intake of nutrients of children with autism and neurotypical controls.

	ASD (n = 25) mean ± SD	Controls (n = 31) mean ± SD	DRI for age
Age (years)	8.9 ± 2.3	8.5 ± 2.6
Total Calories (kcal)	1547 ± 295.5	1584 ± 253.4	1745–1840
Macronutrients
Fat (g)	41.2 ± 12.3	46.4 ± 12.6	Non-determined
MUFA (g)	7.4 ± 3.5	10.3 ± 4.4*
Protein (g)	57.9 ± 11.8	56.2 ± 11.2	19–34
Carbohydrates (g)	243.4 ± 57.9	240.1 ± 45.9	130
Total Dietary Fiber (g)	10.4 ± 4.0	12.1 ± 3.4	25–31
Vitamins
Vitamin A RAE (µg)	652.9 ± 136	406.9 ± 243.4	400–600
Vitamin E Alpha Tocopherol (mg)	2.2 ± 1.02	2.4 ± 1.1	7–11
Vitamin D (µg)	1.6 ± 1.3	2.1 ± 2.7	15
Vitamin B2 Riboflavin (mg)	1.02 ± 0.52	1.09 ± 0.5	0.6–0.9
Vitamin B3 Niacin-equivalents (mg)	15.1 ± 7	17.6 ± 6	8–12
Vitamin B6 (mg)	1.3 ± 0.6	1.4 ± 0.5	0.6–1
Vitamin B12 Cobalamin (µg)	3.6 ± 7.2	2.6 ± 2.1	1.2–1.8
Folate (µg)	152.8 ± 76.4	199.4 ± 243.4	200–300
Vitamin C (mg)	117.9 ± 71.9	86.6 ± 1.1	25–45
Minerals
Calcium (mg)	674.3 ± 363.6	711.6 ± 298.0	1000–1300
Iodine (µg)	34.2 ± 21.6	30.9 ± 14.1	90–120
Iron (mg)	10.3 ± 4.9	11.9 ± 5.4	10–18
Magnesium (mg)	168.7 ± 52.6	166.4 ± 39.2	130–240
Phosphorus (mg)	648.1 ± 210.9	666.7 ± 132.7	500–1250
Potassium (mg)	1554 ± 638.1	1542 ± 478.3	3800–4500
Sodium (mg)	2312 ± 744.6	1999 ± 550.6	1200–1500
Zinc (mg)	5.4 ± 1.7	6.0 ± 1.5	5–8

ASD Autism spectrum disorder; MUFA Monounsaturated fatty acids; DRI Dietary Reference Intakes.

Data are expressed as mean ±SD; *p < 0.05. Data were analyzed using ANOVA.

Table 4. Food habits and gastrointestinal symptomatology of children with autism and neurotypical controls.

	ASD n (%)	Control n (%)	p-value
Food aversion
Yes	22(88.0)	21(60.0)	0.033
No	3(12.0)	14(40.0)
Intolerance to foods
Yes	9(36.0)	1(2.9)	0.006
No	16(64.0)	34(97.1)
Gastrointestinal symptoms
Constipation
Yes	13(52.0)	7(20.0)	0.039
No	12(48.0)	28(80.0)
Diarrhea
Yes	10(40.0)	7(20.0)	0.145
No	15(60.0)	28(80.0)
Other gastrointestinal problems
Yes	10(41.7)	5(16.7)	0.066
No	14(58.3)	25(83.3)
Repetitive behavior related to gastrointestinal problems
Yes	7(29.2)	1(4.8)	0.055
No	17(70.8)	20(95.2)
Depression related to gastrointestinal problems
Yes	5(20.8)	0(0.0)	0.050
No	19(79.2)	22(100.0)

ASD Autism spectrum disorder.

Food aversion = children did not accept specific foods.

Intolerance to food = unpleasant reaction to foods.

Data are expressed as a number of children with the corresponding percentage.

Table 5. Comparison of serum cytokine concentrations of children with autism and neurotypical controls.

Cytokines	ASD	Control	p-value
**IL-6 (pg/mL)	9.30 ± 18.01 (n = 23)	9.92 ± 24.16 (n = 33)	0.539
**IFN-♥ (pg/mL)	23.98 ± 18.44 (n = 22)	13.07 ± 19.29 (n = 33)	0.059
*TGF-→1 (ng/mL)	31.10 ± 13.08 (n = 23)	23.59 ± 10.52 (n = 34)	0.03
**MCP-1 (pg/mL)	34.56 ± 25.66 (n = 22)	30.44 ± 37.46 (n = 33)	0.363

*Data are presented as mean ± SD p-value based on ANOVA.

**Data are presented as median ± interquartile range, p-values are based on Wilcoxon test.

IR Interquartile range; n = indicates the number of studied samples with valid observations.

Figure 1. Alpha Diversity comparison of observed OTUs between ASD and control children. Numbers of observed OTUs on the Y axis and multiple rarefied sequences depth on the X axis. Statistical differences using t-test (P<0.001).

Figure 2. Beta diversity comparison between ASD (red dots) and control children (blue dots) using unweighted UNIFRAC bi-dimensional plot. Statistical differences using MANOVA (P<0.001).

Figure 3. Relative abundance comparison using Gneiss metrics of statistically significant OTUs. Orange bars represent the higher abundant OTUs (including their taxonomic assignation) on the ASD group whilst green bars show the most abundant on the control group. Standard deviation bars are showed.