J Clin Med Res

Journal of Clinical Medicine Research, ISSN 1918-3003 print, 1918-3011 online, Open Access

Article copyright, the authors; Journal compilation copyright, J Clin Med Res and Elmer Press Inc

Journal website https://jocmr.elmerjournals.com

Original Article

Volume 18, Number 10, October 2026, pages 759-769

Targeted Quantitative Polymerase Chain Reaction Analysis of Gut Microbiota and Its Association With Feeding and Mealtime Behavior and Intestinal Permeability in Children With Autism Spectrum Disorder

↓ Figure 1. Fecal zonulin (a) and fecal calprotectin (b) concentrations in children with ASD versus neurotypical controls (mean ± SD; ***P < 0.001). ASD: autism spectrum disorder; SD: standard deviation.

↓ Figure 2. Scatterplots of Total BAMBI score against gut bacterial taxon abundance (Log₁₀ CFU/g) within the ASD group, one panel per taxon, each showing individual children’s data. points and a linear trend line. Spearman’s r and significance are reported per panel (*P < 0.05, **P < 0.01, ***P < 0.001, ns: not significant); correlations that were significant at the uncorrected α = 0.05 level but did not survive Bonferroni correction for the 11 correlations tested (α = 0.0045) are labeled accordingly. ASD: autism spectrum disorder; BAMBI: Brief Autism Mealtime Behavior Inventory; CFU: colony-forming units.

↓ **Table 1.** Demographic and Perinatal Characteristics of the Study Groups
Variable	ASD (N = 70)	Control (N = 70)	Test statistic	P value
Percentages are within-group column percentages and may not sum to 100 due to rounding. M: mean; SD: standard deviation; NICU: neonatal intensive care unit; ASD: autism spectrum disorder.
Age, years, M (SD)	8.2 (2.3)	7.9 (2.2)	t = 0.72	0.472
Sex, n (%)
Male	27 (38.6)	29 (41.4)	χ² = 0.03	0.863
Female	43 (61.4)	41 (58.6)
Residence, n (%)
Urban	37 (52.9)	38 (54.3)	χ² = 0.00	1.000
Rural	33 (47.1)	32 (45.7)
Family income, n (%)
Enough	25 (35.7)	21 (30.0)	χ² = 0.86	0.650
Enough and saving	23 (32.9)	28 (40.0)
Not enough	22 (31.4)	21 (30.0)
Father’s education, n (%)
Basic/none	23 (32.9)	28 (40.0)	χ² = 2.37	0.306
Primary/secondary	30 (42.9)	32 (45.7)
University	17 (24.3)	10 (14.3)
Mother’s education, n (%)
Basic/none	27 (38.6)	28 (40.0)	χ² = 1.06	0.589
Primary/secondary	32 (45.7)	27 (38.6)
University	11 (15.7)	15 (21.4)
Parental smoking, n (%)
Yes	40 (57.1)	35 (50.0)	χ² = 0.46	0.498
No	30 (42.9)	35 (50.0)
Siblings, n, M (SD)	1.7 (1.5)	1.7 (1.4)	t = 0.00	1.000
Low birth weight, n (%)
Yes	43 (61.4)	30 (42.9)	χ² = 4.12	0.042
No	27 (38.6)	40 (57.1)
Full-term delivery, n (%)
Yes	38 (54.3)	47 (67.1)	χ² = 1.92	0.166
No	32 (45.7)	23 (32.9)
NICU admission, n (%)
Yes	37 (52.9)	38 (54.3)	χ² = 0.00	1.000
No	33 (47.1)	32 (45.7)
Weight-for-age z-score, M (SD)	0.4 (2.0)	−0.1 (1.9)	t = 1.53	0.129
Height-for-age z-score, M (SD)	0.1 (2.0)	0.1 (2.1)	t = −0.01	0.993

↓ Table 1. Demographic and Perinatal Characteristics of the Study Groups

Variable

ASD (N = 70)

Control (N = 70)

Test statistic

P value

Percentages are within-group column percentages and may not sum to 100 due to rounding. M: mean; SD: standard deviation; NICU: neonatal intensive care unit; ASD: autism spectrum disorder.

Age, years, M (SD)

8.2 (2.3)

7.9 (2.2)

t = 0.72

0.472

Sex, n (%)

Male

27 (38.6)

29 (41.4)

χ² = 0.03

0.863

Female

43 (61.4)

41 (58.6)

Residence, n (%)

Urban

37 (52.9)

38 (54.3)

χ² = 0.00

1.000

Rural

33 (47.1)

32 (45.7)

Family income, n (%)

Enough

25 (35.7)

21 (30.0)

χ² = 0.86

0.650

Enough and saving

23 (32.9)

28 (40.0)

Not enough

22 (31.4)

21 (30.0)

Father’s education, n (%)

Basic/none

23 (32.9)

28 (40.0)

χ² = 2.37

0.306

Primary/secondary

30 (42.9)

32 (45.7)

University

17 (24.3)

10 (14.3)

Mother’s education, n (%)

Basic/none

27 (38.6)

28 (40.0)

χ² = 1.06

0.589

Primary/secondary

32 (45.7)

27 (38.6)

University

11 (15.7)

15 (21.4)

Parental smoking, n (%)

Yes

40 (57.1)

35 (50.0)

χ² = 0.46

0.498

30 (42.9)

35 (50.0)

Siblings, n, M (SD)

1.7 (1.5)

1.7 (1.4)

t = 0.00

1.000

Low birth weight, n (%)

Yes

43 (61.4)

30 (42.9)

χ² = 4.12

0.042

27 (38.6)

40 (57.1)

Full-term delivery, n (%)

Yes

38 (54.3)

47 (67.1)

χ² = 1.92

0.166

32 (45.7)

23 (32.9)

NICU admission, n (%)

Yes

37 (52.9)

38 (54.3)

χ² = 0.00

1.000

33 (47.1)

32 (45.7)

Weight-for-age z-score, M (SD)

0.4 (2.0)

−0.1 (1.9)

t = 1.53

0.129

Height-for-age z-score, M (SD)

0.1 (2.0)

0.1 (2.1)

t = −0.01

0.993

↓ **Table 2.** Fecal Biomarkers of Intestinal Permeability and Inflammation
Biomarker	ASD (N = 70)	Control (N = 70)	Test statistic (t)	P value
SD: standard deviation; ASD: autism spectrum disorder.
Fecal zonulin, ng/mL (mean ± SD)	133.5 ± 25.6	59.8 ± 17.5	t = 19.84	< 0.001
Fecal calprotectin, µg/g (mean ± SD)	151.1 ± 42.2	61.8 ± 24.1	t = 15.39	< 0.001

↓ Table 2. Fecal Biomarkers of Intestinal Permeability and Inflammation

Biomarker

ASD (N = 70)

Control (N = 70)

Test statistic (t)

P value

SD: standard deviation; ASD: autism spectrum disorder.

Fecal zonulin, ng/mL (mean ± SD)

133.5 ± 25.6

59.8 ± 17.5

t = 19.84

< 0.001

Fecal calprotectin, µg/g (mean ± SD)

151.1 ± 42.2

61.8 ± 24.1

t = 15.39

< 0.001

↓ **Table 3.** Gut Bacterial Taxa: Detection Rate and Abundance (Log₁₀ CFU/g) in ASD versus Control Groups
Taxon	Detected, ASD N (%)	Detected, control N (%)	Detection χ², P	Abundance*, Log₁₀ CFU/g (ASD vs. control), t, P
*Among taxon-positive samples only (n varies by taxon; see Section “Gut microbiota composition” text and Methods).
Prevotella	38 (54.3)	65 (92.9)	χ² = 24.83, P < 0.001	5.5 ± 0.8 vs. 7.8 ± 0.7; t = −14.35, P < 0.001
Bifidobacterium	32 (45.7)	59 (84.3)	χ² = 21.22, P < 0.001	5.4 ± 0.8 vs. 7.7 ± 0.8; t = −14.00, P < 0.001
Lactobacillus	30 (42.9)	68 (97.1)	χ² = 46.56, P < 0.001	5.7 ± 0.7 vs. 7.7 ± 0.7; t = −12.49, P < 0.001
Clostridium difficile	51 (72.9)	34 (48.6)	χ² = 7.67, P = 0.006	7.5 ± 0.9 vs. 4.0 ± 0.6; t = 21.10, P < 0.001
Bacteroidetes	36 (51.4)	26 (37.1)	χ² = 2.34, P = 0.126	7.4 ± 1.0 vs. 4.1 ± 0.7; t = 15.72, P < 0.001
Firmicutes	38 (54.3)	53 (75.7)	χ² = 6.15, P = 0.013	7.5 ± 1.1 vs. 4.3 ± 0.6; t = 16.30, P < 0.001

↓ Table 3. Gut Bacterial Taxa: Detection Rate and Abundance (Log₁₀ CFU/g) in ASD versus Control Groups

Taxon

Detected, ASD N (%)

Detected, control N (%)

Detection χ², P

Abundance*, Log₁₀ CFU/g (ASD vs. control), t, P

*Among taxon-positive samples only (n varies by taxon; see Section “Gut microbiota composition” text and Methods).

Prevotella

38 (54.3)

65 (92.9)

χ² = 24.83, P < 0.001

5.5 ± 0.8 vs. 7.8 ± 0.7; t = −14.35, P < 0.001

Bifidobacterium

32 (45.7)

59 (84.3)

χ² = 21.22, P < 0.001

5.4 ± 0.8 vs. 7.7 ± 0.8; t = −14.00, P < 0.001

Lactobacillus

30 (42.9)

68 (97.1)

χ² = 46.56, P < 0.001

5.7 ± 0.7 vs. 7.7 ± 0.7; t = −12.49, P < 0.001

Clostridium difficile

51 (72.9)

34 (48.6)

χ² = 7.67, P = 0.006

7.5 ± 0.9 vs. 4.0 ± 0.6; t = 21.10, P < 0.001

Bacteroidetes

36 (51.4)

26 (37.1)

χ² = 2.34, P = 0.126

7.4 ± 1.0 vs. 4.1 ± 0.7; t = 15.72, P < 0.001

Firmicutes

38 (54.3)

53 (75.7)

χ² = 6.15, P = 0.013

7.5 ± 1.1 vs. 4.3 ± 0.6; t = 16.30, P < 0.001

↓ **Table 4.** Autism Severity and Mealtime/Feeding Behavior (BAMBI) by Group
Variable	ASD (N = 70)	Control (N = 70)	Test statistic (t)	P value
CARS: Childhood Autism Rating Scale; BAMBI: Brief Autism Mealtime Behavior Inventory; SD: standard deviation; ASD: autism spectrum disorder.
CARS total score (mean ± SD)	39.5 ± 5.7
Age at diagnosis, years (mean ± SD)	4.0 ± 1.2
Total BAMBI score (mean ± SD)	54.1 ± 6.3	54.7 ± 5.5	t = −0.60	0.547
Food refusal subscale (mean ± SD)	15.1 ± 3.6	15.4 ± 3.1	t = −0.57	0.566
Limited food variety subscale (mean ± SD)	23.6 ± 4.1	23.9 ± 3.7	t = −0.33	0.745
Autism-specific mealtime behavior subscale (mean ± SD)	15.4 ± 3.5	15.5 ± 2.8	t = −0.11	0.915

↓ Table 4. Autism Severity and Mealtime/Feeding Behavior (BAMBI) by Group

Variable

ASD (N = 70)

Control (N = 70)

Test statistic (t)

P value

CARS: Childhood Autism Rating Scale; BAMBI: Brief Autism Mealtime Behavior Inventory; SD: standard deviation; ASD: autism spectrum disorder.

CARS total score (mean ± SD)

39.5 ± 5.7

Age at diagnosis, years (mean ± SD)

4.0 ± 1.2

Total BAMBI score (mean ± SD)

54.1 ± 6.3

54.7 ± 5.5

t = −0.60

0.547

Food refusal subscale (mean ± SD)

15.1 ± 3.6

15.4 ± 3.1

t = −0.57

0.566

Limited food variety subscale (mean ± SD)

23.6 ± 4.1

23.9 ± 3.7

t = −0.33

0.745

Autism-specific mealtime behavior subscale (mean ± SD)

15.4 ± 3.5

15.5 ± 2.8

t = −0.11

0.915

↓ **Table 5.** Spearman’s Correlations Within the ASD Group (N = 70, or as Noted)
Variables compared	Spearman's r	P value	N
ASD: autism spectrum disorder; CARS: Childhood Autism Rating Scale; BAMBI: Brief Autism Mealtime Behavior Inventory.
CARS vs. fecal zonulin	0.03	0.815	70
CARS vs. fecal calprotectin	−0.06	0.618	70
Total BAMBI vs. fecal zonulin	−0.04	0.754	70
Total BAMBI vs. fecal calprotectin	0.03	0.806	70
CARS vs. total BAMBI	−0.04	0.724	70
Total BAMBI vs. Prevotella abundance	−0.35	0.029	38
Total BAMBI vs. Bifidobacterium abundance	−0.68	< 0.001	32
Total BAMBI vs. Lactobacillus abundance	−0.41	0.023	30
Total BAMBI vs. Clostridium difficile abundance	0.74	< 0.001	51
Total BAMBI vs. Bacteroidetes abundance	0.63	< 0.001	36
Total BAMBI vs. Firmicutes abundance	0.20	0.237	38
CARS vs. any of the six taxa	−0.10 to 0.23	All > 0.16	30–51

↓ Table 5. Spearman’s Correlations Within the ASD Group (N = 70, or as Noted)

Variables compared

Spearman's r

P value

ASD: autism spectrum disorder; CARS: Childhood Autism Rating Scale; BAMBI: Brief Autism Mealtime Behavior Inventory.

CARS vs. fecal zonulin

0.03

0.815

CARS vs. fecal calprotectin

−0.06

0.618

Total BAMBI vs. fecal zonulin

−0.04

0.754

Total BAMBI vs. fecal calprotectin

0.03

0.806

CARS vs. total BAMBI

−0.04

0.724

Total BAMBI vs. Prevotella abundance

−0.35

0.029

Total BAMBI vs. Bifidobacterium abundance

−0.68

< 0.001

Total BAMBI vs. Lactobacillus abundance

−0.41

0.023

Total BAMBI vs. Clostridium difficile abundance

0.74

< 0.001

Total BAMBI vs. Bacteroidetes abundance

0.63

< 0.001

Total BAMBI vs. Firmicutes abundance

0.20

0.237

CARS vs. any of the six taxa

−0.10 to 0.23

All > 0.16

30–51

↓ **Table 6.** Multiple Linear Regression Predicting Total BAMBI Score From *Bifidobacterium* and *Clostridium difficile* Abundance (N = 22)
Predictor	β (unstandardized)	t	P value
Model R² = 0.584, adjusted R² = 0.540, F(2, 19) = 13.35, P < 0.001. CFU: colony-forming units.
Constant	44.09	4.06	0.001
Bifidobacterium, Log₁₀ CFU/g	−2.67	−2.38	0.028
Clostridium difficile, Log₁₀ CFU/g	3.32	3.36	0.003

↓ Table 6. Multiple Linear Regression Predicting Total BAMBI Score From Bifidobacterium and Clostridium difficile Abundance (N = 22)

Predictor

β (unstandardized)

P value

Model R² = 0.584, adjusted R² = 0.540, F(2, 19) = 13.35, P < 0.001. CFU: colony-forming units.

Constant

44.09

4.06

0.001

Bifidobacterium, Log₁₀ CFU/g

−2.67

−2.38

0.028

Clostridium difficile, Log₁₀ CFU/g

3.32

3.36

0.003