Table 3 Effect of water, sanitation, handwashing, and nutrition intervention on stress response and DNA methylation measurements on Bangladeshi children at age 28 months

From: A cluster-randomized trial of water, sanitation, handwashing and nutritional interventions on stress and epigenetic programming

Outcome	N	Absolute Mean	Mean	SD	Unadjusted difference: Intervention v. Control		Age- and sex-adjusted difference: Intervention v. Control		Full adjusted difference: Intervention v. Control^a
					95% CI	P-value	95% CI	P-value	95% CI	P-value
Ln pre-stressor salivary alpha-amylase (U/ml)
Control	354	74.9	4.01	0.81
Nutrition + WSH	394	75.92	3.98	0.89	−0.03 (−0.19, 0.13)	0.731	−0.02 (−0.18, 0.14)	0.785	−0.02 (−0.17, 0.14)	0.834
Ln post-stressor salivary alpha-amylase (U/ml)
Control	339	124.06	4.47	0.9
Nutrition + WSH	375	122.53	4.41	0.99	−0.06 (−0.26, 0.13)	0.530	−0.06 (−0.24, 0.13)	0.550	−0.01 (−0.2, 0.18)	0.928
Slope between pre- and post-stressor salivary alpha-amylase (U/ml/min)
Control	335	2.99	2.99	6.43
Nutrition + WSH	367	2.73	2.73	5.29	−0.22 (−1.07, 0.64)	0.622	−0.2 (−1.02, 0.62)	0.635	0.09 (−0.72, 0.91)	0.821
Residualized gain score for alpha-amylase (U/ml)
Control	335	0.77	0.77	108.21
Nutrition + WSH	368	−0.7	−0.7	89.31	−1.17 (−15.16, 12.82)	0.870	−0.19 (−13.82, 13.45)	0.979	4.97 (−8.79, 18.72)	0.479
Ln pre-stressor salivary cortisol (µg/dl)
Control	357	0.17	−2.08	0.69
Nutrition + WSH	396	0.18	−2.03	0.73	0.05 (−0.08, 0.18)	0.458	0.04 (−0.09, 0.17)	0.553	0.04 (−0.08, 0.17)	0.487
Ln post-stressor salivary cortisol (µg/dl)
Control	312	0.34	−1.49	0.96
Nutrition + WSH	385	0.42	−1.26	0.95	0.24 (0.07, 0.4)	0.005	0.21 (0.05, 0.37)	0.010	0.21 (0.06, 0.36)	0.007
Slope between pre- and post-stressor cortisol (µg/dl/min)
Control	311	0.01	0.01	0.01
Nutrition + WSH	380	0.01	0.01	0.01	0.002 (0, 0.003)	0.035	0.002 (0, 0.003)	0.053	0.002 (0, 0.003)	0.031
Residualized gain score for cortisol (µg/dl)
Control	311	−0.03	−0.03	0.27
Nutrition + WSH	380	0.03	0.03	0.32	0.06 (0.01, 0.12)	0.023	0.06 (0, 0.11)	0.033	0.06 (0.01, 0.11)	0.018
Mean arterial pressure (mmHg)
Control	353	65.18	65.18	6.14
Nutrition + WSH	399	65.5	65.5	6.78	0.33 (−1, 1.66)	0.625	0.32 (−1.06, 1.7)	0.649	0.31 (−0.84, 1.46)	0.596
Resting heart rate (bpm)
Control	358	109.49	109.49	14.43
Nutrition + WSH	398	108.12	108.12	17.12	−1.35 (−4.08, 1.39)	0.334	−1.43 (−4.14, 1.27)	0.299	−1.65 (−4.26, 0.97)	0.218
Logit-transformed NR3C1 exon 1F promoter methylation
Control	346	0.39	−3.53	0.09
Nutrition + WSH	396	0.38	−3.53	0.1	−0.001 (−0.02, 0.018)	0.917	−0.001 (−0.021, 0.019)	0.895	−0.003 (−0.02, 0.014)	0.720
Logit-transformed NGFI-A transcription factor binding site methylation
Control	336	0.9	−3.39	0.25
Nutrition + WSH	386	0.77	−3.43	0.26	−0.04 (−0.08, 0)	0.037	−0.04 (−0.08, 0)	0.080	−0.04 (−0.08, 0)	0.037

We used targeted maximum likelihood estimation with influence curve-based standard errors accounting for clustered observations from the trial’s geographic block-randomized design. Confidence intervals were adjusted for clustered observations using robust standard errors. The statistical tests were two-sided, and no adjustments were made for multiple comparisons.
^aAdjusted for prespecified covariates: child sex, child birth order, mother’s age, mother’s height, mother’s education, number of children <18 years in the household, number of individuals living in the compound, distance in minutes to the primary water source, household food security, household floor materials, household wall materials, household electricity, and household assets (wardrobe, table, chair, clock, khat, chouki, radio, television, refrigerator, bicycle, motorcycle, sewing machine, mobile phone, cattle, goats, and chickens), child age at dates of urine, vitals, and saliva collection, and monsoon season at dates of urine, vitals, and saliva collection. For salivary alpha-amylase and cortisol analyses only, time of sampling was included as a covariate.
Source data are provided as a Source Data file.

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Table 3 Effect of water, sanitation, handwashing, and nutrition intervention on stress response and DNA methylation measurements on Bangladeshi children at age 28 months

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