Zimbabwe Environmental Enteropathy

and Child Growth Study

Jean Humphrey, Sc.D.ZVITAMBO HIV Care and Prevention for Mothers and Children in Zimbabwe

Johns Hopkins Bloomberg School of Public HealthEBF Tozodii Collaboration between Zimbabwe MoHCW and ZVITAMBO

Why and How measure EE to evaluate WASH?

• Background to Zimbabwe trial: (WHY measure EE?)

• Objectives and design of our trial

• HOW are we and other groups planning to measure EE?

Child stunting undernutrition:Magnitude

Of the 555 million childrenin developing countries:

•32% stunted

Consequences

• >20% of all <5 year mortality

• Fewer years of school and poorer performance while there

• Long-term cognitive defects

• Lower adult economic productivity

• For girls: increased risk of stunted children.

Source: Victora CG, de Onis M, Hallal PC, Blössner M, Shrimpton R. Worldwide timing of growth faltering: revisiting implications for interventions using the World Health Organization growth standards. Pediatrics, 2010 (Feb 15 Epub ahead of print)

Mean height for age z-scores by age by region

Stunting prevalence and number affected in developing countries

1990 2000 2010

010

2030

4050

Stu

ntin

g (%

)

40.3

48.6

23.7

39.337.7

18.1

38.2

27.6

13.5

1990 2000 2010

050

100

150

200

Num

ber o

f stu

nted

(milli

ons)

45

190

13

51

138

10

60

100

7

AFRICA ASIA LATIN AMERICASource: Department of Nutrition, World Health Organization

Infant DietsReview of 42 infant feeding

studies/programs published 1996-2006:(Dewey and Adu-Afarwuah, 2008)

Most studies produced statistically significant growth effects:

Children who received interventions gained

•0-760 g more weight (0.0 – 0.76 WAZ)

•0 – 1.7 cm taller (0.0 – 0.64 LAZ)

At best, diet solved 1/3 of the problem

The very best of these interventions produced a 0.7 Z-score improvement

BUT, the average growth deficit of African and Asian children is –2.0 Z-score

Diarrhea associated with stunting

Diarrhea accounted for 5-20% of linear growth deficit in some studies:

Guatemala: 10% (Martorell, 1975) Mexico: 5% (Condon, 1977) Gambia: 20% (Rowland, 1977) Bangladesh: 20% (Black, 1984) Peru: 16% (Checkley, 2003)

Year x(SD) LAZ 12 months x(SD) LAZ 24 months

1979 -1.3 (1.0) -2.0 (0.9)1993 -1.7 (1.0) -2.1 (0.9)

Lancet Nutrition Series (2008):Role of Sanitation and Hygiene

in undernutrition:

Implement Sanitation & Hygiene interventions

with 99% coverage

Reduce Diarrhea by 30%

Reduce undernutrition

by 2.4%

However:

Evidence exists that the effect of WASH interventions on linear growth is independent of its effect on diarrhea.

DHS data: 17,000 children, 8 countriesExamined child diarrhea and growth across levels of improving sanitation and water.

Growth was a MORE sensitive indicator of sanitation and water benefits than was diarrhea

Improved WASH associated with ~0.1-0.6 increased HAZ (similar effect to diet interventions)

Peru: (Checkley, et al)Cohort followed from birth to 24 months

Measured growth and diarrheaAscertained sanitation and water

At 24 mths, average child was 2.4 cm shorter than normal:

16% of the deficit (0.4 cm/2.4 cm) explained by diarrhea

40% of the deficit (1.0 cm/2.4 cm) explained by poor sanitation and water

(equivalent to about ~0.8 HAZ)

Hypothesis• A major (if not primary) cause of child

undernutrition is a subclinical condition of small intestine:

Tropical Enteropathy (TE)Environmental Enteropathy (EE)

In addition, we hypothesize:

• EE can be reduced or prevented with improved WASH

• Major causal pathway from poor sanitation/hygiene to stunting is NOT diarrhea, but is EE.

Poor Sanitation

and HygieneChild

undernutrition

EE

Diarrhea

If true:• Lancet series substantially underestimated

the contribution of sanitation/hygiene to child growth because the impact was modeled entirely through clinical diarrhea incidence.

• Given attributable mortality and long-term consequences of stunting:

WASH has been undervalued since it has been appraised primarily for its impact on diarrhea.

What is EE?

Flat, inflamed gut, characterized by:»villous atrophy»modest malabsorption»inflammatory cell infiltrate»increased permeability

NormalEnvironmental enteropathy

What causes EE?

• Nearly every paper concludes it is poor sanitation and hygiene, but there is little direct evidence.

EE results from unrestrained stimulation of gut T-cells

• Hyperstimulation of human gut T-cells rapidly leads to villous atrophy, crypt hyperplasia, inflammatory cell infiltrate

• So, high concentrations of bacteria colonize or cause repeated infections in the small intestine and induce the morphologic changes of EE through a T-cell mediated process.

• Mechanism recently described for EE – (Veitch, 2001; Campbell, 2003)

• Same mechanism in other IBDs

• Diarrhea Caused by pathogenic bacteria

• EEEven non-pathogenic bacteria may be involved: :

in germ-free animals, commensal bacteria in low concentrations exerts a trophic effect on intestinal epithelium; morphology shifts from “supranormal” (ie, very tall villi) to normal. If true -challenging implications for WASH interventions.

Child growth studiesDunn Nutrition Laboratory, The Gambia

1985 - 2005

• Growth faltered between 3 – 14 months of age

• Not explained by inadequate diet or corrected by dietary supplementation

• Not associated with diarrhea

• Was associated with indicators of EE

WAZ: – 0.5 – 2.4

LAZ: – 0.5 – 1.8

Lactulose:Mannitol Gambian: 0.15 0.40

UK: 0.15 0.10

Lactulose:Mannitol (gut permeability)

(Campbell, J Nutr 2003)

Plasma immunoglobulin concentrations rose to 60-200% above normal between 3 and 9 months of age

IgG

IgA

IgM

Plasma immunoglobulins (immune stress)(Campbell, J Nutr 2003)

EndoCAb: Endotoxin is a component of gram neg bacteria. Rise in its specific antibody indicates translocation of endotoxin across gut wall.

Differences in EndoCAb explained 46% of linear growth

Endotoxin Core Antibody

(Campbell, J Nutr 2003)

Growth and Intestinal Enteropathy in Gambian Children (Campbell, J Nutr 2003)

r 2 with height growth:

IgG: 0.43

L:M: 0.22

EndoCAb: 0.46

Together, IgG, L:M and EndoCAb explained 56% of linear growth

Strongest = EndoCAb

• Children had diarrhea 7.3% of the time, but had L:M values associated with growth suppression 76% of the time.

• Diarrhea represented only one-tenth of the growth-suppressive intestinal disease these children endured most of their lives.

Diarrhea: tip of the enteric disease iceberg?

Diarrhea

Dirty Chicken Study

Raised in cages tht were steam-cleaned

Clean ChickDirty ChickRaised in ‘usual’ chicken cages filled with feces, dust, and dander

Dirty Chicken Study•‘Clean Chicks’ grew better than ‘Dirty Chicks’

•‘Dirty Chicks’ fed antibiotics grew as well as ‘Clean Chicks’

•Antibiotics did not improve growth in ‘Clean Chicks’

•Poor growth in ‘Dirty Chicks’ accompanied by elevated plasma IL-1 (indicator of immune stress)

•Elevated IL-1 not observed in ‘Clean Chicks’ or ‘Dirty Chicks’ fed antibiotics

When confronted with incessant microbial challenge:

•Gambian infants and dirty chicks mount an immune response

•Immune Stress diverts nutrients

•AWAY from growth

•TOWARD the business of fighting an infection:

• making immunoglobulins, acute phase proteins, cytokines, and

•glycolysis to fuel increased energy requirements

Proposed Trial

Primary Aim

To evaluate the independent and combined effects of:

Improved Sanitation/Hygiene

Improved Infant Diet

on:

Growth birth to 18 months

Anemia at 18 months

Four Program Inputs Across entire area of both districts

1. Repair public boreholes to remove barrier of water quantity

2. Strengthen VHW network3. Promote EBF4. Provide high quality PMTCT

Trial Design

• NOT a program evaluation or trial of interventions that could be scaled up immediately

• IS a Proof of Concept Study because no research or program has achieved better than ~ 0.7 shift in LAZ.

• So for WASH arm, research question is: If we interupt fecal-oral transmission in infants

(birth - 18 mo) will linear growth improve?

• So our focus is on:• Identifying THE prime pathways for fecal-oral

transmission specifically for infantsTh b st i b h i h t hi th hi h st

Trial DesignSo our focus is:

• Identifying THE prime pathways for fecal-oral transmission specifically for infants

• Applying the very best in behavior change to achieve the highest and most sustained uptake possible

• Provision of tools (latrine they want to use, HW facility, soap, periennial access to water, Water Guard) to facilitate improved behaviors

• Attaining and maintaining a high degree of constrast between intervention groups

Trial Design

• 2x2 factorial– Sanitation/Hygiene , Infant feeding, Both, Standard Care

• Cluster-randomized– 120 clusters each the catchment area of 2 VHWs

• Pregnant women recruited (probably through pregnancy surveillance), infants followed to 18 mo– 4100 HIV-negative and 700 HIV-positive

• Most interventions delivered by supported district health team

• Latrines constructed by project staff and families under EHT and project supervision

• Outcomes measured by research staff

3 Years Formative Research• Infant feeding, including specific for HIV-

exposed

• HH WASH practices, facilitators and constraints (high demand for Blair VIP latrines – cost the only constraint)

• Primary vectors (observational, microbiologial) for transmission among infants: their own hands, sibling and mother hands, soil, chicken feces

Standard Care1. Repaired boreholes2.ReRevived VHW network3. Strengthened PMTCT care4. EBF Campaign

Nutrition1. Standard care

2. VHW promotes optimal use of local foods for complementary feeding

3. 20 g Nutributter daily provided for infants (6-18 mo)

Sanitation/Hygiene1. Standard care

2. VIP latrine in all HH’s of enrolled pregnant women.

3. VHW promotes safe fecesdisposal, HWWS after fecal contract, water treatment, hygienic infant food preparation. (RETHINKING!)

Nutrition & Sanitation/Hygiene

1. Standard care2. All Nutrition interventions3. All Sanitation/Hygiene

interventions

Intervention Arms

VHW trainingAfter clusters are randomized, VHWs will receive additional

special training on 8-10 modules developed for each intervention:

• Standard of Care (key maternal-neonatal interventions in standard MoHCW curriculum; eg: EBF, seek early ANC, EPI)

• Infant feeding

• WASH

Similar modes, intensity, creativity, entertainment (eg, 1 short video, 1 drama, 1 hand-up reminder, 1 game, etc)

Same number of family visits

Assessing Outcomes: Primary outcomes

ALL infants :

1. Weight and Length at birth and 3-monthly

2. Hemoglobin at 18 months by Hemacue3. Comprehensive baseline survey for HH

and community factors4. Coverage and uptake of Standard Care

interventions

EE:the hypothesized pathway between WASH and growth

How will we measure EE?

Domains of EE

1. Crypt hyperplasia, villous atrophy = reduced small intestinal surface area

2. Inflammation3. Increased permeability4. Microbial translocation5. Systemic immune activation6. Hormonal response to IA7. Linear growth

Intestinal surface area

Measure of enterocyte mass

Inflammation

Zvitambo:• Fecal calprotectin or mRNA calprotectin

MalEd • Fecal alpha-1-antitrypson• Fecal Lactoferrin (confounded by BM) • (+several other markers on their

piloting list)

Intestinal permeability

• L:M sugar challenge urine test

Strong consensus this remains the gold standard test.

Microbial Translocation

• Endotoxin Core Antibody- Campbell and Zvitambo data

To pilot• 16s ribosomal bacterial DNA (MalEd

is also piloting viral DNA) in whole blood by real-time quantitative PCR on whole blood

Ln EndoCAb according to LAZ at 18 mo

• Case-Control; ZVITAMBO archived specimens from non-HIV-exposed infants at 18 months, matched on BW (n=65 per group)

Multivariate model, two variables retained:• EndoCAb >40 MU/L 2.95 (1.27 – 6.83) p < 0.01• Years maternal schooling 0.74 (0.58 – 0.94) p < 0.02

Group Ln EndoCAb (IQR) % Ln EndoCAb >40 MU/LControls 43.1 (35.6 – 52.2) 49.2 %Stunted 70.2 (57.0 – 86.4) 75.4%

Immune Activation

• Total plasma IgG

• Acute phase proteins: CRP, AGP

• Proinflammatory cytokines: IL-1, IL-6,TNF-α

• Plasma sol CD14 (specific marker of immune response to LPS exposure; esp strong evidence in HIV literature)

Hormonal response to IA

• IGF-1Depressed in other IBD’s in childrenDirectly upregulated by IL-6

TeamMoHCWGoldberg Mangwadu (Co-PI), plus directorates of nursing and nutritionZvitamboJean Humphrey (Co-PI) Mduduzi Mbuya, Naume Tavengwa, Kuda

Mutasa, Robert NtoziniJohns Hopkins Bloomberg School of Public HealthLarry Moulton, Jim Tielsch (J Humphrey)CornellRebecca Stoltzfus, David Sahn, Steve YoungerUCLAndrew PrendergastLondon School Hygiene Trop MedSandy Cairncross, Val CurtisMcGillAmee Manges