Effects of single and combined water, sanitation and handwashing

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Characterization of Natural and Affected Environments

Effects of single and combined water, sanitation and handwashing interventions on fecal contamination in the domestic environment: a cluster-randomized controlled trial in rural Bangladesh Ayse Ercumen, Andrew Mertens, Benjamin F. Arnold, Jade Benjamin-Chung, Alan E. Hubbard, Mir Alvee Ahmed, Mir Himayet Kabir, Md. Masudur Rahman Khalil, Ashish Kumar, Md. Sajjadur Rahman, Sarker Masud Parvez, Leanne Unicomb, Mahbubur Rahman, Pavani Ram, Thomas F. Clasen, Stephen P. Luby, and John M. Colford Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b05153 • Publication Date (Web): 26 Sep 2018 Downloaded from http://pubs.acs.org on September 27, 2018

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Environmental Science & Technology

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Effects of single and combined water, sanitation and handwashing interventions on fecal contamination in the domestic environment: a cluster-randomized controlled trial in rural Bangladesh Ayse Ercumen1,2*, Andrew Mertens2, Benjamin F. Arnold2, Jade Benjamin-Chung2, Alan E. Hubbard2, Mir Alvee Ahmed3, Mir Himayet Kabir3, Md. Masudur Rahman Khalil3, Ashish Kumar3, Md. Sajjadur Rahman3, Sarker Masud Parvez3, Leanne Unicomb3, Mahbubur Rahman3, Pavani K. Ram4, Thomas Clasen5, Stephen P. Luby6, John M. Colford Jr.1 1. Department of Forestry and Environmental Resources, North Carolina State University, Raleigh, NC, 27695 2. School of Public Health, University of California, Berkeley, CA, 94720 3. Infectious Disease Division, International Centre for Diarrhoeal Disease Research, Bangladesh, Dhaka, 1212, Bangladesh 4. University at Buffalo, Buffalo, NY, 14214 5. Rollins School of Public Health, Emory University, Atlanta, GA, 30322 6. Infectious Diseases & Geographic Medicine, Stanford University, Stanford, CA, 94305 * Corresponding author: Ayse Ercumen Jordan Hall, 2800 Faucette Drive, Raleigh, NC 27695 [email protected] Word count Abstract: 198 (limit 200) Text: 4900 Tables: 0 Figures: 3 x 300 + 2 x 600 = 2100 Total including tables and figures: 7000 (limit 7000)


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Abstract

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Water, sanitation and hygiene interventions have varying effectiveness in reducing fecal

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contamination in the domestic environment; delivering them in combination could yield

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synergies. We conducted environmental assessments within a randomized controlled trial

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in Bangladesh that implemented single and combined water treatment, sanitation,

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handwashing (WSH) and nutrition interventions (WASH Benefits, NCT01590095). After

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one and two years of intervention, we quantified fecal indicator bacteria in samples of

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drinking water (from source or storage), child hands, children’s food and sentinel objects.

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In households receiving single water treatment interventions, E. coli prevalence in stored

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drinking water was reduced by 50% and concentration by 1-log. E. coli prevalence in food

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was reduced by 30% and concentration by 0.5-log in households receiving single water

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treatment and handwashing interventions. Combined WSH did not reduce fecal

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contamination more effectively than its components. Interventions did not reduce E. coli in

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groundwater, on child hands and on objects. These findings suggest that WSH

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improvements reduced contamination along the direct transmission pathways of stored

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water and food but not along indirect upstream pathways. Our findings support

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implementing water treatment and handwashing to reduce fecal exposure through water

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and food but provide no evidence that combining interventions further reduces exposure.

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Background

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Diarrheal disease transmission occurs through a complex web of environmentally

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mediated pathways including drinking water, food, hands, fomites and vectors. The

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complexity arises from the multitude of transmission routes, broad diversity of pathogens,

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importance of environmental conditions, and interactions between the environment and

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human behavior. Treating drinking water before consumption and washing hands with

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water and soap lower fecal contamination of drinking water and hands, respectively, and

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reduce reported diarrhea 1–4. However, hands are rapidly re-contaminated by contact with

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objects and surfaces 5–7 and treated water can be re-contaminated by hands and utensils

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during storage 8. Sanitation interventions target reducing contamination further upstream

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by isolating fecal matter from the ambient surroundings but sanitation improvements to

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date have generally not reduced fecal indicators in the environment and shown mixed

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impact on health 9–12.

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Combining water, sanitation and hygiene improvements could yield synergistic benefits.

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Coupling water treatment and safe storage with a hygiene intervention could reduce re-

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contamination of treated water through cleaner hands, while combining handwashing with

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sanitation improvements could reduce re-contamination of washed hands through reduced

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exposure to fecal matter in the environment. Water, sanitation, and hygiene interventions

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could also work in concert to reduce food contamination by making treated water available

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for food preparation, facilitating handwashing before food handling and reducing breeding

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sites for flies transmitting pathogens to stored food 13,14. No studies have investigated


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whether combined water, sanitation and hygiene interventions more effectively reduce

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contamination of drinking water, food, hands and fomites than individual interventions. We

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conducted an environmental assessment within a randomized controlled trial in

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Bangladesh (WASH Benefits, ClinicalTrials.gov NCT01590095) to assess whether (1) water,

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sanitation and hygiene interventions vs. controls and (2) combined vs. single water,

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sanitation and hygiene interventions reduce fecal indicator bacteria and flies in the

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domestic environment.

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Methods

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Study design

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WASH Benefits enrolled participants in four districts (Gazipur, Kishoreganj, Mymensingh,

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Tangail) in rural Bangladesh. These areas were selected because their groundwater

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chemistry was suitable for the trial’s chlorine-based water treatment intervention 15 and

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because they had no other major ongoing or planned water, sanitation and hygiene

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programs. The trial enrolled pregnant women identified by screening the study area. Using

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global positioning system (GPS) coordinates, eight adjacent eligible women were grouped

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into clusters, and each eight clusters formed a block. Clusters were block-randomized into

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study arms by an off-site investigator (BFA), providing pair-matched randomization. WASH

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Benefits followed the birth cohort born to the enrolled pregnant women (“index children”)

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for two years. Further details of the trial design have been previously described 16. The

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primary outcomes of WASH Benefits were child diarrhea and growth, and additional

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outcomes included protozoan and soil-transmitted helminth (STH) infections; these have


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been reported separately 17–19. Measures of environmental contamination were pre-

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specified intermediate outcomes 16.

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The trial had six intervention arms including single and combined water, sanitation,

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handwashing and nutrition interventions and a double-sized control arm receiving no

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intervention. The intervention arms included (1) water treatment: point-of-use water

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treatment with sodium dichloroisocyanurate (NaDCC, Aquatabs) (Medentech, Wexford,

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Ireland) and safe storage in a narrow-mouth, lidded container with spigot, (2) sanitation

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improvements: upgrades to concrete-lined double-pit latrines, and provision of child

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potties and scoops for feces disposal, (3) handwashing promotion: provision of

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handwashing stations in the kitchen and latrine areas with a water reservoir, a bottle of

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soapy water mixture and a basin for rinse water (4) nutrition improvements: provision of

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lipid-based nutrient supplements for children aged 6-24 months, recommendations for

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exclusive breastfeeding for children aged up to 6 months and age-appropriate nutrition

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recommendations from pregnancy through 24 months of age, (5) combined water,

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sanitation and handwashing (WSH), and (6) nutrition plus combined WSH (N+WSH).

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Community health promoters hired from among local women and trained specifically for

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the study visited intervention households six times per month on average to demonstrate

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and encourage the targeted behaviors (e.g., water treatment, handwashing at critical times,

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latrine use for defecation) and supply intervention products for free throughout the trial

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period. Control households did not receive any interventions or health promoter visits.

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User adherence to the interventions was measured through spot-check and structured


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observations in unannounced household visits (as an independent investigation from the

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study activities reported here) 20. These demonstrated high adherence throughout the trial,

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including hardware availability observed by spot-checks and user practices assessed by

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structured observations. Spot-checks indicated >95% of households in arms receiving the

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sanitation intervention had a latrine with a water seal compared to 23% of controls, and

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>77% of households in arms receiving the handwashing intervention had water and soap

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present 94% of households in sanitation arms adults used a hygienic

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latrine for defecation (vs. 40% of controls), in >67% of households in handwashing arms

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participants washed hands with soap after using the latrine (vs. 29% of controls) and in

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>65% of households in water treatment arms participants drank chlorine-treated water

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from a safe storage container (vs. none of controls) 20. Further details of user adherence

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have been described 20–22.

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Procedures

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A subset of trial participants in the control, nutrition, WSH and N+WSH arms was enrolled

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in an environmental enteric dysfunction (EED) substudy. EED enrollment was based on

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proximity to the laboratory and therefore (unlike the parent trial) not geographically pair-

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matched. We conducted an environmental assessment among EED substudy participants.

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This allowed us to assess the environmental impact of the combined WSH intervention by

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comparing pooled data from the WSH and N+WSH arms (both receiving the WSH package,

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referred to as “WSH arm” hereinafter) to pooled data from the control and nutrition arms

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(neither one receiving the WSH intervention, referred to as “control arm” hereinafter). We


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also sampled a random subset of households in the single water and handwashing arms to

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assess the impact of combined vs. individual interventions. The sanitation arm was not

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sampled as a previous assessment showed no early environmental impact in this arm 23

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and a separate longitudinal study is underway to assess the long-term effect of sanitation

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on environmental contamination (manuscript forthcoming). Sampling was conducted at

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two timepoints, approximately 1 and 2 years after intervention initiation, to match the

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timing of the trial’s health outcome measurements (see SI Text S1 and Figure S1 for details

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of all environmental assessments nested within WASH Benefits).

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Trained field workers from the International Centre for Diarrhoeal Disease Research,

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Bangladesh (icddr,b) visited households enrolled in the environmental assessment to

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collect samples. In the control and WSH arms, we quantified fecal indicator bacteria in

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drinking water, on index child hands, on toy ball “sentinel objects” and (in year two only) in

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food served to young children (Fig 1). Sentinel objects are pre-sterilized objects (e.g., toy

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ball) left in the household to take up contamination from the domestic environment while

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household members interact with it and then tested for fecal indicator bacteria 24,25. They

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serve as a measure of overall contamination of domestic surfaces and objects and have

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been shown to distinguish households with vs. without improved water, sanitation and

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hygiene conditions in Bangladesh 26,27. In single intervention arms, we only collected a

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relevant subset of sample types expected to be directly affected by the interventions; we

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sampled drinking water and (in year two) children’s food in the water arm, and index child

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hands and (in year two) children’s food in the handwashing arm. Additionally, field staff

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examined caregiver and index child hands (fingernails, fingerpads and palms) in the


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control, handwashing and WSH arms for visible dirt; observed dirt on hands has been

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validated as a proxy for handwashing 28 and shown to correlate with bacterial counts on

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hands 29. Field staff also enumerated and speciated synanthropic flies in the kitchen and

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latrine areas in the control and WSH arms.

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Sample collection. Samples were collected using sterile Whirlpak bags (Nasco Modesto,

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Salida, CA). Clean gloves were worn while collecting hand rinse, toy rinse and food samples.

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To collect drinking water, field workers asked the respondent to provide a glass of water in

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the same manner they would give it to their children and pour approximately 150 mL into

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the Whirlpak; field workers recorded if the water was provided from the source (tubewell)

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or from a storage container. If the respondent reported using chlorine, sodium thiosulfate

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was used to neutralize residual chlorine, and an additional sample was collected to

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measure the free chlorine residual with a digital colorimeter (Hach Pocket Colorimeter II).

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To sample child hands, field workers asked the respondent to place the index child’s left

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hand into a Whirlpak pre-filled with 250 mL of distilled water. The hand was massaged

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from outside the bag for 15 seconds and shaken for 15 seconds. The procedure was

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repeated with the right hand in the same bag, and the rinse water was preserved in

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Whirlpak. To sample sentinel objects, field workers delivered pre-sterilized non-porous

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plastic toy balls to participants and instructed them to let the index child as well as other

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children in the compound play freely with the ball. They returned 24 hrs later to rinse the

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ball in a Whirlpak pre-filled with 250 mL of distilled water by massaging it from outside the

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bag and shaking the bag, for 15 seconds each. The rinse water was preserved for analysis

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and the toy was left at the household; a new ball was delivered at the time of the year two


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sampling. To sample food, field workers identified stored food prepared to be served to

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children 200 colonies for water, hand rinses and toy rinses and >500 colonies for food were

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classified as too numerous to count (TNTC). The higher detection limit for the food samples

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reflects the larger surface area of the 100-mm plates, allowing a higher number of colonies

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to be visually distinguished (see Table S1 for detection limits for each sample type).

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Quality control. One laboratory control per analyst per day and 5% replicates (repeat

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aliquots from same Whirlpak for every 20th sample) were processed. Field workers

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collected 10% field blanks (one blank for every 10 samples) by asking respondents to pour

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distilled water from a sterile bottle into a Whirlpak as if collecting a water sample, by

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opening and massaging a pre-filled Whirlpak as if sampling a hand, and by rinsing a pre-

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sterilized toy ball in pre-filled Whirlpak as if sampling a toy.

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Fly counts. To enumerate flies, field workers identified a suitable location in the kitchen and

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latrine areas (away from the stove smoke, under a roof or protected from rain) and

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horizontally hung three 1.5-foot strips of non-baited sticky fly tape. They returned to the

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household 24 hrs later to count the captured synanthropic flies and speciate them

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according to a visual identification chart 33.

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Ethics


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Participants provided written informed consent in the local language (Bengali). The study

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protocol was approved by human subjects committees at the icddr,b (PR-11063),

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University of California, Berkeley (2011-09-3652), and Stanford University (25863).

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Statistical methods

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We pre-specified and registered our analysis plan on Open Science Framework

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(https://osf.io/6u7cn/).

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Sample size. The EED substudy, within which the environmental sampling was nested,

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targeted 1500 households (375/arm) and enrolled approximately 2000 households in year

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one to allow for attrition by year two. In addition, we enrolled 180 water arm households

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and 360 handwashing arm households in the environmental assessment. We obtained

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measures of contamination and intra-class correlation coefficients from the literature and

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unpublished pilot data (see analysis plan). We used a one-sided α of 0.05, assuming that

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the interventions would decrease but not increase contamination 1,2. Our sample size

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yielded 80% power to detect a 0.20 log10 reduction in E. coli concentration in drinking

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water, hands and toy rinses at each sampling timepoint, compared to controls.

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Parameters of interest. Our outcomes were (1) prevalence and concentration of E. coli and

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total/fecal coliforms, (2) prevalence and number of flies near the kitchen and latrine, and

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(3) prevalence of caregiver and child hands with visible dirt. Our parameters were

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prevalence ratios (PR) and differences (PD) for binary measures, log10 reductions for E. coli

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and coliform concentrations, and fly count ratios for the number of flies. We substituted


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bacterial counts with half the lower detection limit for non-detects, 200 CFU for TNTC

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water, hand rinse and toy rinse samples and 500 CFU for TNTC food samples 34. Means for

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multiple dilutions were obtained by dividing the sum of plate counts by the total sample

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volume filtered for all countable plates.

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Estimation strategy. We compared all intervention arms to controls and the combined WSH

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arm to the single water and handwashing arms. Analyses were intention-to-treat; this

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preserves the randomization and is appropriate given the high intervention adherence 20.

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Randomization balanced covariates across arms 17. Therefore, we relied on unadjusted

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estimates in our analysis. We estimated unadjusted parameters using generalized linear

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models with robust standard errors, and a log link for PRs, linear link for PDs and log10

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reductions, and log link allowing for over-dispersion (negative binomial regression) for fly

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count ratios. Secondary analyses adjusted for pre-specified covariates using doubly-robust

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targeted maximum likelihood estimation (TMLE) incorporating an ensemble machine

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learning method called Super Learner 35,36. We conducted separate comparisons at each of

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the two measurement rounds and performed a Bonferroni correction for multiple

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measurements by multiplying the p-values for effect estimates by two. We conducted

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separate analyses stratifying by whether the sampled drinking water came from the source

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or storage container. We assessed effect modification by season by including an interaction

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term for wet vs. dry season; Bangladesh has a monsoon season (Jun-Oct), during which it

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receives >80% of its rain 37 and environmental contamination levels typically increase 38.

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Results and Discussion


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Enrollment

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Of 2445 households selected for EED enrollment in year one, we enrolled 1980 (81%) in

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the environmental assessment between October 2013 and December 2014; we also

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enrolled 181 water arm households and 366 handwashing arm households. At year two,

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among 1515 households successfully enrolled in the EED substudy, we enrolled 1363

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(90%) in the environmental assessment between May 2015 and May 2016; we also

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enrolled 184 water arm households and 356 handwashing arm households. Reasons for

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households not being enrolled were no live birth or index child death (10% in year one,

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0.3% in year two), absence or relocation (7% in year one, 4% in year two) and refusal (2%

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in year one, 5% in year two). Covariates were balanced between arms among enrolled

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households (Table S2).

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Observed hand cleanliness

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Among caregivers in the control arm, dirt was observed on 80% of nails and 33-34% of

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fingerpads and palms at year one, and 50% of nails and 15% of fingerpads and palms at

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year two (Figs 2-3). Among children in the control arm, dirt was observed on 89% of nails

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and 62-66% of fingerpads and palms at year one (mean child age: 14 months), and 66% of

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nails and 42-43% of fingerpads and palms at year two (mean child age: 30 months); these

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ages roughly coincided with WHO windows for rolling, crawling and learning to walk (5-18

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months) vs. walking well (>18 months) 39. Caregivers in the handwashing arm were

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substantially less likely to have dirt on fingerpads and palms but not under fingernails at

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both timepoints (Figs 2-3). Similarly, children in the handwashing arm were less likely to


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have dirt on fingerpads and palms and, to a smaller extent, under fingernails (Figs 2-3).

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Caregivers or children in the WSH arm were no less likely to have dirt on their hands than

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controls at either timepoint (Figs 2-3).

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Fecal contamination

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Levels of contamination. We analyzed 6409 samples at year one and 6127 at year two. Of

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drinking water samples, 29% were provided directly from a tubewell and 71% from a

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storage container (in arms receiving the water intervention, this was predominantly the

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provided safe storage container). Among controls, we detected E. coli in 59-62% of

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tubewells, 90-93% of stored drinking water, 96-98% of child hands and 76-81% of toys at

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the two timepoints and 55% of children’s food at year two (Figs 4-5).

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Intervention effects. Among households receiving water treatment interventions, 90% of

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stored drinking water samples were reported to be chlorinated, and of these, 80% had

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detectable (>0.1 mg/L) free chlorine residual at the two timepoints, while 1% of controls

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reported water treatment. Compared to controls, stored drinking water in the single water

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arm had approximately 50% reduction in E. coli prevalence and 1-log reduction in E. coli

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concentration at both timepoints (Yr1: prevalence ratio [PR]=0.53 (0.44, 0.63), ∆log10 = -

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0.96 (-1.13, -0.80); Yr2: PR=0.51 (0.43, 0.62), ∆log10 = -1.05 (-1.23, -0.88); all Bonferroni-

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corrected p

Effects of single and combined water, sanitation and handwashing

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