Environmental Enteropathy Pilot StudyCleaner households are associated with improved

gut function in Bangladesh

Audrie Lin, PhDBen Arnold, PhDSadia Afreen, MPHJack Colford, MD, MPH, PhDSteve Luby, MDWASH Benefits Team

3 August 2013

Photo: Audrie Lin

555 million preschool children in developing countries: 32% stunted & 20% underweight

WHO 2011, Humphrey 2009, Lunn 1991, Campbell 2003, Bhutta 2008

Vitamin A & Zinc supplementation

Protein supplementation

Complementary feeding

Breastfeeding promotion

Micronutrient supplementation in pregnancy

Decrease stunting at 36 months by

36%

Increased Child Morbidity & Mortality Poor Cognitive Development Reduced Adult Economic Productivity

Inadequate Diet

Undernutrition & Growth Faltering

555 million preschool children in developing countries: 32% stunted & 20% underweight

WHO 2011, Humphrey 2009, Checkley 2008

Increased Child Morbidity & Mortality Poor Cognitive Development Reduced Adult Economic Productivity

Poor Sanitation & Hygiene

Diarrhea

Inadequate Diet

Undernutrition & Growth Faltering

25% of all stunting in 24-month-old children

attributable to having ≥ 5 diarrheal episodes in first 2

years of life

Proposed Causal Pathway of Environmental Enteropathy

555 million preschool children in developing countries: 32% stunted & 20% underweight

WHO 2011, Humphrey 2009, Lunn 1991, Campbell 2003

Environmental Enteropathy

??

Increased Child Morbidity & Mortality Poor Cognitive Development Reduced Adult Economic Productivity

Poor Sanitation & Hygiene

Diarrhea

Inadequate Diet

Undernutrition & Growth Faltering

Small Intestine Has Large Absorptive Area

Absorptive surface area of small intestine = 250 m2

Mara 2009

Healthy Intestine Environmental Enteropathy

What is Environmental Enteropathy (EE)?

• Healthy villi • Absorb nutrients• Pathogen barrier

• Villous atrophy• Malabsorption• “Leaky” intestine

Inflammatory disorder of small intestine that reduces nutrient absorption and increases intestinal permeability

Lactulose Mannitol Diagnostic Assay for EE

Lactulose sugar– Too large to be absorbed by healthy intestine– L is a measure of barrier efficacy– EE lactulose absorption

Mannitol sugar– Small and moves across intestine in quantities

proportional to absorptive surface area– M is a measure of absorptive capacity– EE mannitol absorption

Healthy Intestine = Low L:M ratio EE = High L:M ratio

LM Dose

5-Hour Urine Collection

Measure L:M in Urine

Adapted by Christine Stewart from Victora et al., 2010

Critical Period For Growth Faltering

Increase in Lactulose:Mannitol Ratios with Age in Gambian Village Children

1-3 3-6 6-9 9-12 12-15

Age (Months) Lunn 1991

The Gambia

United Kingdom

EE Background Studies

• In Bangladesh, L:M ratio increases from age 3 – 12 months in a rural subdistrict (Goto et al Br J Nutr 2009; Trans Roy Soc of Trop

Med Hyg 2009)

• Enteropathy develops in people who move from more developed countries to more contaminated environments (e.g., Peace Corps volunteers). (Lindenbaum et al. Ann Intern Med 1966)

• Within 17 months after the Peace Corps volunteers’ return to New York City, the biopsies normalized and within 1 to 35 months, most of the volunteers had normal, functioning small intestines. (Lindenbaum et al. Ann Intern Med 1971)

Photo : throbule www.panoeramio.com

Study Question

Is environmental enteropathy a potential mechanism to explain the association between environmental contamination and stunting?

SHEWA-B EvaluationSanitation Hygiene Education and Water Supply, Bangladesh

• Government of Bangladesh / UNICEF program

• Targeting 20 million people• icddr,b contracted with the

evaluation– 50 intervention and 50 matched

control clusters

– Clusters randomly selected

– 10 households per cluster

– Monthly follow up for 2 years

Would Children in Relatively Clean Households Have Less EE Than Children in Dirtier Households?

Dirty Clean

5.4% 6.4%

SHEWA-B WASH Survey• 993 households • July 2007 – Oct 2009

WASH Benefits?

Definitions and Household Selection Criteria

Clean

• flush/septic/piped sewerage or pit latrine with slab & water seal

• [E. coli] < 10 CFU/100 ml

• handwashing station w/ water and soap

Dirty

• open defecation, hanging toilet, open pit latrine, pit latrine with slab but no water seal, or flush/pour to “somewhere else”

• [E. coli] ≥ 10 CFU/100 ml

• No handwashing station or handwashing station w/o water and/or w/o soap

Photo: Flickr zafarbaig08

Illustrations: practicalaction.org

Finding the study children

• SHEWA-B evaluation– 993 households– July 2007 – Oct 2009

• Environmental enteropathy assessment– May 2010– 136 children– 49 sub-districts

Ages: 8 – 48 months

Data Collection

1 Stool Sample

1 Urine Sample

1 Finger prick Blood Sample

• Soil Transmitted Helminths• Giardia lamblia• Cryptosporidium parvum• Entamoeba histolytica

Anthropometry Data

• IgG endotoxin core antibodies • Total IgG

• Dose of lactulose-mannitol solution• 5 Hour urine collection• L:M absorption ratio

Household WASH Survey

• Weight• Length/Height• Head circumference

• Drinking water source

• Toilet facility• Handwashing station

Household Characteristics   Clean Dirty P-value (n=67) (n=55)

Female 0.54 0.58 0.62

Age in months at enrollment 10.5 12.5 0.39

Have electricity 72% 36% <0.01

Own

radio 30% 16% 0.08

b/w television 31% 15% 0.04

color television 24% 5% 0.01

refrigerator 7% 0 0.02

# of people living in the household 5.91 6.31 0.47

# of rooms 2.94 1.8 <0.01

earth/bamboo floor 69% 93% 0.001

Clean Households Were Wealthier

Clean Households Were Objectively Cleaner But Not Radically Different From Dirty Households

Measure Clean Dirty P

stool visible on slab or floor 22% 72% <0.01

child < 5 yr feces disposal in no specific place/bush/field

78% 81% 0.67

fingernails of the child are clean 20% 10% 0.12

palms of the child are clean 42% 21% 0.03

finger pads of the child are clean 42% 17% 0.01

fingernails of the respondent are clean 37% 13% <0.01

palms of the respondent are clean 67% 56% 0.29

finger pads of the respondent are clean 66% 55% 0.28

Note: These indicators were not used to categorize households

Children in Clean Households Were 0.9 SDs Taller

Mean HAZ(2010)

Stunting %(2010)

Dirty –2.57 74%

Clean –1.66 33%

Difference 0.91 40%

This effect is 3x the magnitude seen in many nutritional interventions

---Height-for-Age Z-score (HAZ)

Severe Stunted

Clean (N=66) Dirty (N=53)

HAZ was a primary outcome in the WASH Benefits Pilot

Children in Clean Households Were Taller and Less Likely to be Stunted

Mean age (mo) 9 35 9 35

HAZ Diff 95% CI P R2

Unadjusted 0.91 ( 0.17, 1.65) <0.001 0.12

Adjusted for age + sex 0.96 ( 0.51, 1.41) <0.001 0.15

Fully Adjusted * 0.54 ( 0.06, 1.01) 0.028 0.31

* Adjusted for age, sex, housing materials, household electricity, household size, land ownership, household head occupation, durable good ownership (bike, tables, chairs, beds, radio, tv).

Children From Clean Households Have Higher HAZ (+0.54) After Adjustment

Children < 5 Years in Clean Households Have Lower Ascaris Infection Prevalence

Parasite infection was a secondary outcome in the WASH Benefits Pilot

Environmenal Enteropathy BiomarkersChildren in Clean Households had:

Inflammation(Total IgG)

Dirty

Antibody Response(EndoCAb)

CleanDirty

Better Gut Function(LM Ratio)

CleanDirtyCleanLn

L:M

Rati

o

Ln E

ndoC

Ab M

U/m

l

Ln T

otal

IgG

mg/

ml

LM Ratio Levels in Context

Campbell et al. 2003

UK Infants

Clean Environment

Dirty Environment

Rural Bangladeshi children with somewhat– Cleaner water– Better toilets– Better equipped

handwashing stations

Had– Less environmental

enteropathy– Less parasitic

infection– Better growth

?WASH Benefits

Conclusion: Cleaner Households were Associated with Improved Gut Function in Bangladesh

Photo: ICDDR,B

Cross-sectional Study

WASH Benefits Main Trial

WASH Benefits Hypotheses

Improvements in: – Water quality (WA)– Sanitation (S)– Hygiene (H)– Nutrition – WASH – WASH + Nutrition

Less diarrhea Fewer parasites Less environmental

enteropathy Improved child

growth Improved child

development

Objective: Measure the effect of water, sanitation, handwashing, and nutrition interventions on child health, growth and development

WASHB Main Trial Overview of the Design

6 treatment arms + a double-sized control

Kenya WASH Interventions

Chlorine dispensers at water sources

Water Quality

Improved Tippy Taps + soap provision

Handwashing

Sani-scoop Child Potties Improved Latrines+ +

Sanitation

Bangladesh WASH Interventions

Safe StorageAquatabs (NaDCC) +

(www.aquatabs.com)

Soapy water dispensers + soap provision

Sani-scoop Child Potties Dual Pit Latrines+ +

Water Quality

Sanitation

Handwashing

Bangladesh & Kenya Nutrition Interventions

NutritionPromotion

Daily LNS(6 – 24 mos)

+

Exclusive breastfeeding through 6 months

Continued breastfeeding with LNS (6 – 24 months)

Encourage preparation of micronutrient-dense food

Feed child at least 2-3 times per day (6-8 mos) and 3-4 times per day (9-24 mos)

Next-generation Nutributter (iLiNS project)

(Draws heavily from Alive & Thrive)

In a subsample of 1,500 children in each cohort, we will collect urine, blood, and stool specimens to measure EE biomarkers

WASHB Main Trial EE Subsample

Bangladesh & Kenya Sample Types and Assays

Stool

Urine

Venous Blood

• Alpha 1-Antitrypsin • Myeloperoxidase • Neopterin • Regenerating Protein 1?• Calprotectin?• Giardia lamblia • Cryptosporidium parvum • Entamoeba histolytica

• Total IgG • Micronutrients, Lead, Genetics• C-Reactive Protein? • EndoCAb?• IFAB-P?• IL-6?• Zonulin?• Citrulline?

• Lactulose-Mannitol

PBMCs (Bangladesh)

• Immunologic Assays

Acknowledgments

Field Research OfficerMd. Mamun-Ar-Rashid

Field Team Leaders:Mahadi HasanSyeda Luthfa FamidaShanzida TaslinAshish Kumar KunduAyesha BegumSamsul HaqueShafi Md. Ali Siddik

ICDDR,BSadia AfreenSania AshrafLeanne UnicombRashidul HaqueTahmeed AhmedRubhana RaqibTarique Md. Nurul HudaSheuli (FRO)FellowsAdministrationA. M. Shamsir AhmedVirology LabHematology Lab Nutritional Biochemistry Lab Atiya Sharmeen & the MAL-ED Clinic

Stanford UC BerkeleySteve Luby Ben Arnold

Jack Colford

ShireeRie GotoNicholas Mascie-TaylorMasud Rana

Field Research AssistantsShabina YesminLubna TaniTania ZahirMd. Shafiqul HaiderIrin AkterMd. AlimojjamanFatema Begum SumiNurul AlamMahmuda AkterShadia AhmedTowhida AkterAfsana Binte BariRahela Akter

Field AssistantsMd. AlamMd. Mohasin KabirMd. Saiful IslamMd. Jamilur RahmanMd. Shah AlamMd. Aminul IslamMd. Junayed Hossain

Extra Slides

Geography of Environmental Enteropathy

50 – 95% incidence in developing countries McKay 2010, Menzies 1999

Correlation of EE with GDP Per Capita

Menzies 1999

GDP correlates with absorptive capacity GDP correlates with intestinal permeability

Variation in Lactulose:Mannitol Ratio With Age in The Gambia

La

ctu

lose

:Man

nit

ol P

erm

ea

bili

ty R

ati

o

Age Group (Years)Campbell 2002

The Gambia

United Kingdom

Children from Clean Households are 22 percentage points less likely to be stunted

DiffStunting

95% CI P

Unadjusted –0.40 (–0.57, –0.24) <0.001

Adjusted for age + sex –0.42 (–0.59, –0.26) <0.001

Fully Adjusted * –0.22 (–0.42, –0.02) 0.030

Robustness Checks

Mahalanobis matched** –0.25 (–0.46, –0.04) 0.019

GenMatch** –0.28 (–0.50, –0.05) 0.017

* Adjusted for age, sex, housing materials, household electricity, household size, land ownership, household head occupation, durable good ownership (bike, tables, chairs, beds, radio, tv).

** Matching estimators include the adjustment covariates used in fully adjusted models, plus the propensity score estimated using main effects of the covariates.

Enteropathy biomarkersclean versus dirty

Biomarker * Clean Dirty Difference

Mean Mean in SDs 95% CI

Ln Total IgG –0.14 0.16 –0.30 (–0.69, 0.08)

Ln EndoCAb –0.12 0.16 –0.29 (–0.64, 0.07)

Ln L:M Ratio –0.19 0.23 –0.42 (–0.77, –0.07)

* Biomarkers standardized by subtracting the sample mean and dividing by the sample SD.

Enteropathy biomarkersclean versus dirty

Biomarker Clean Dirty

  Mean Mean

Total IgG (mg/ml) 28.19 38.77

EndoCAb (MU/ml) 50.02 62.42

L:M Ratio 0.21 0.31

Lactulose (mg/100 ml) 134 207

Mannitol (mg/100 ml) 689 637

EndoCAb Levels in ContextCampbell et al. 2003

UK InfantsClean Env.Dirty Env.