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Letter

Occurrence of host-associated fecal markers on child hands, household soil, and drinking water in rural Bangladeshi households Alexandria B. Boehm, Dan Wang, Ayse Ercumen, Meghan Shea, Angela Harris, Orin C. Shanks, Catherine Kelty, Alvee Ahmed, Zahid Hayat Mahmud, Benjamin Arnold, Claire Chase, Craig Kullmann, John Colford, Stephen Luby, and Amy J Pickering Environ. Sci. Technol. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.estlett.6b00382 • Publication Date (Web): 13 Oct 2016 Downloaded from http://pubs.acs.org on October 14, 2016

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Environmental Science & Technology Letters

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Occurrence Of Host-Associated Fecal Markers On Child Hands, Household Soil, And Drinking

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Water In Rural Bangladeshi Households

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Alexandria B. Boehm1, Dan Wang1, Ayse Ercumen2, Meghan Shea1, Angela R. Harris1, Orin C.

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Shanks3, Catherine Kelty3, Alvee Ahmed4, Zahid Hayat Mahmud 4, Benjamin F. Arnold2, Claire

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Chase5, Craig Kullmann5, John M. Colford, Jr.2, Stephen P. Luby6, Amy J. Pickering*1,6

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1. Department of Civil and Environmental Engineering, Stanford University, Stanford, CA

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94305

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2. Division of Epidemiology, University of California, Berkeley, Berkeley, CA, 94720

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3. USEPA Office of Research & Development, Cincinnati, OH, 45268

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4. International Center for Diarrheal Diseases Research, Bangladesh, Dhaka 1000, Bangladesh

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5. The World Bank, Washington, D.C.. 20433

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6. Woods Institute for the Environment, Stanford University, Stanford, CA, 94305

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* Corresponding author: Email: [email protected], Tel: +1 510 410 2666

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ACS Paragon Plus Environment

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Abstract

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We evaluated whether provision and promotion of improved sanitation hardware (toilets and

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child feces management tools) reduced rotavirus and human fecal contamination of drinking

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water, child hands, and soil among rural Bangladeshi compounds enrolled in a cluster-

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randomized trial. We also measured host-associated genetic markers of ruminant and avian feces.

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We found evidence of widespread ruminant and avian fecal contamination in the compound

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environment; non-human fecal marker occurrence scaled with animal ownership. Strategies to

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control non-human fecal waste should be considered when designing interventions to reduce

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exposure to fecal contamination in low-income settings. Detection of a human-associated fecal

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marker and rotavirus was rare and unchanged by provision and promotion of improved sanitation

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to intervention compounds. The sanitation intervention reduced ruminant fecal contamination in

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drinking water and general (non-host specific) fecal contamination in soil, but overall had limited

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effects on reducing fecal contamination in the household environment.

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Introduction

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Lack of sanitation access in low-income rural settings has been linked to diarrheal illness1 and

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impaired growth in children under five years old.2 Recent evidence has also connected unsanitary

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living conditions to child environmental enteropathy.3

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Microbial contamination on hands,4–10 water,7,11–13 soil,14,15 and household floors4 has been

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documented in areas with poor sanitation. However, the majority of studies has measured

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microbial contamination using fecal indicator bacteria (FIB) such as Escherichia coli (EC) and

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enterococci (ENT). FIB are found in feces of many animal hosts and even in natural

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reservoirs.16–18 There are far fewer studies that measure microbial contamination using enteric

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pathogens or host-associated genetic markers of fecal contamination.

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Previous intervention trials have investigated whether provision of sanitation hardware has

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improved water quality and hand contamination as measured by FIB.2,19,20 The results of those

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studies have been equivocal with most showing no change in contamination, which could

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indicate limited effects of the sanitation intervention on environmental contamination (although

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two of these trials had low rates of latrine adoption). Another possible explanation for the

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equivocal results is that the outcomes of interest (FIB) are not primarily from human feces, but

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rather are from animal feces.21 In this case, proper disposal of human feces through latrine

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provision may not necessarily reduce overall FIB contamination.

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This study evaluated whether provision and promotion of improved sanitation to rural

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Bangladeshi compounds reduced the incidence of a human-associated fecal genetic marker and


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rotavirus RNA in stored drinking water, courtyard soil, and on child hands. In addition, we

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assessed the occurrence of ruminant and avian-associated fecal genetic markers to infer whether

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animals contribute to microbial contamination in the domestic environment.

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Materials and Methods

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Environmental sample collection was nested within the WASH Benefits trial in rural

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Bangladesh,22 a randomized controlled trial designed to measure the effect of improved water

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quality, sanitation, handwashing, and nutritional interventions on child diarrhea and growth. We

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collected environmental samples (stored drinking water, soil, child hand rinse) from a subset of

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compounds in the control and sanitation arms of the study; 497 compounds (249 from the control

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arm and 248 from the sanitation arm) were included.

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A compound consisted of between 3-10 households comprised of typically blood relatives, with

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a shared courtyard. Each compound had at least one child under five years of age. Groups of

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adjacent compounds were assigned into clusters, and clusters were randomly assigned into the

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sanitation versus control arm within geographical strata. In sanitation arm compounds, each

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household lacking a hygienic latrine was provided with a concrete ring-based dual pit latrine that

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had a slab, water seal, and a superstructure for privacy. The sanitation intervention also included

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a potty for young children and a metal scoop for removal of child and animal feces from the

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environment and their safe disposal in the latrine. A behavior change program encouraged

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regular use of hardware components through weekly compound visits throughout the study. The

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sanitation hardware and behavior intervention were designed after two years of pilot testing with

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documented high user uptake of the selected interventions.22 The control arm received no

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hardware or behavior change intervention.


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Microbial source tracking (MST) marker validation. We conducted a MST marker validation

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study to choose the most sensitive and specific MST markers for use in the study setting. MST

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marker validation has been completed in a handful of large studies,23–26 but local testing is

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recommended before implementation in new geographic settings.24,27

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Fecal samples were collected from 20 chickens, 20 ducks, 20 cows, 20 goats, and 15 humans in

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the study communities. Three to four individual fecal specimens of the same animal species were

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combined at equal masses to form a 2.0 g composite (Table S1); the result was five composite

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samples per species. The composite samples were made into fecal slurries and EC and ENT were

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enumerated in the slurries using defined-substrate assays (IDEXX, Westbrook, MN). For QPCR

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analysis, 2 mL of slurry were filtered through membrane filters seated in disposable filter

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funnels. Further details are in the Supporting Information (SI).

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DNA was extracted from filters using a modified MoBio PowerWater RNA Isolation Kit

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(Carlsbad, CA)5. For each extraction batch (10-20 samples), an extraction blank control was

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included. DNA was used as template in QPCR reactions for three human-associated (BacHum,28

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HumM2,26 HF183Taqman29), three ruminant-associated (BacCow,28 BacR,30 Rum2Bac31), and

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one avian-associated (Avian GFD32) MST markers. These markers were chosen because they

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performed well in previous studies.23,24,33 The sensitivity and specificity of the markers were

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determined using metrics described previously.4,23 The most sensitive and specific host-

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associated MST markers were selected to analyze the environmental samples. (See SI for more

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details).

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Environmental sampling and survey. Environmental sampling occurred from November 2013

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through March 2014. At each enrolled compound, field staff collected a stored drinking water

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sample, a hand rinse from one child under five years old,7 and a soil sample from the

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compound’s courtyard where the youngest child under five years old had most recently played or

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spent time according to a compound resident. Respondents were asked how many ruminant and

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avian (e.g., chickens, ducks, geese) species were owned by the compound.

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Samples were preserved on ice and processing begun within 12 h. Samples were analyzed for EC

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and fecal coliform (FC) using IDEXX defined-substrate assays. Aliquots of the water and hand

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rinse samples were membrane filtered to collect nucleic acids from bacteria and viruses and the

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filters archived using the same technique as for the fecal slurries. In addition, laboratory process

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control blanks were processed (see SI). Soil was archived in centrifuge tubes and stored at -80°C.

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Filters and soil were tested for rotavirus RNA,34 general Bacteroidales DNA (GenBac3),35 as

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well as select host-associated MST markers. GenBac3 targets fecal bacteria in the Bacteriodetes

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class.26,35

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DNA and RNA were co-extracted from the water and hand rinse filters using the same method as

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for the fecal samples. DNA and RNA were co-extracted from soil samples using a protocol

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developed in this study (further described in SI). Bacterial DNA and viral RNA recovery from

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soil using the extraction method is estimated at 50% and 10%, respectively while recovery from

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filters was previously estimated at 7% and 17%, respectively.36

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The nucleic acid extracts from environmental samples were assessed for substances that inhibit

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QPCR using a spike and dilute method;37 results informed the dilution level of extract to run

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during QPCR. All samples were run in duplicate. Each QPCR plate included a standard curve

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run in triplicate as well as triplicate no-template controls. Environmental samples were scored as

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positive if at least one of the two replicates amplified, even if the concentration was below the

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lower limit of quantification (LLOQ). Lowest detectable concentration (LDC) and LLOQ values

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were calculated by converting one copy (cp)/reaction and 10 cp/reaction (the most dilute

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standard that consistently amplified), respectively, to appropriate units (cp/100 mL, cp/2 hands,

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cp/g dry soil). Linear regression using respective instrument run-specific standard data was used

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to estimate molecular marker concentrations. SI contains further details.

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To compare MST marker occurrence between the control and sanitation groups we used

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logistical regression for binary outcomes and linear regression for continuous outcomes; all

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models included indicator variables for each pair matched cluster (minus a reference cluster) as

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well as robust standard errors to account for geographic clustering of observations.22

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Results & Discussion

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Quality assurance and control. HumM2 and Avian-GFD were not detected in no template

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controls, or extraction or laboratory processing blanks. BacR was detected at levels below the

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LLOQ in 2 of 78 extraction blanks (these were co-extracted with hand rinse samples). GenBac3

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was detected in 50% of the extraction blanks and 41 of 111 laboratory processing blanks with

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most of these below or near the LLOQ (see SI). This cross contamination is likely a result of the

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extraordinarily high levels of the GenBac3 present in the samples, and has been observed by


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others23. The LDC and LLOQ values for the environmental samples were 50 and 500 cp/100 mL

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water, 125 and 1250 cp/2 hands, and approximately 400 and 4000 cp/g soil (exact value

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depended on moisture content).

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MST marker validation. We evaluated MST marker sensitivity and specificity using both a

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binary and quantitative assessment approach23 (Figure S1 and Table S2). In brief, the binary

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approach assesses the percent of target and non-target fecal samples where the MST marker was

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and was not detected to calculate the sensitivity and specificity, respectively; a cutoff of 80%

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was used to define good performance.23 The quantitative approach takes into account the

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concentration of marker detected in tested feces and requires higher marker concentrations in

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target versus non-target feces (see SI).

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Of the three tested human-associated MST markers, HumM2 was specific via the quantitative

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assessment approach and sensitive via the binary assessment approach. The other human MST

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markers (HF183 and BacHum) were not specific by either assessment approach. Previous work

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in urban Bangladesh (Dhaka) found that none of these three human-associated markers was

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specific4 and that an assay not tested herein (HF183 SYBR) was sensitive and specific.38 A large

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method evaluation study in the US found that HF183 and HumM2 were sensitive and specific,

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but that BacHum lacked specificity.23 The different outcomes of these validation studies

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underscores the need for local validation before human-associated MST assays are applied to

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environmental samples.27

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Of the three ruminant-associated MST markers, BacR performed the best; it was the only marker

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that was sensitive and specific as determined by both assessment approaches. The high

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specificity of ruminant-associated assays has been observed in multiple geographic settings.24

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In the present study, the avian-associated marker was specific via the quantitative assessment

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approach and sensitive via the binary approach. Tested in the US and Australia, it performed

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with good sensitivity and specificity.32,39 In another study conducted in urban Bangladesh, it was

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neither sensitive nor specific.4

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Given their good sensitivity and specificity in our study area (rural Bangladesh), we used

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HumM2, BacR, and avian-GFD markers to assess the presence of human, ruminant, and avian

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fecal contamination in the environmental samples, respectively.

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Environmental fecal contamination. Across all compounds, EC and FC concentrations were on

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the order of 10 MPN/2 hands, 10 MPN/100 mL of stored water, and 105 MPN/g soil. GenBac3

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concentrations were on the order of 106 cp/2 hands, 104 cp/100 mL water, and 106 cp/g soil, and

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(Tables S3 and S4).

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The number of samples positive for each MST marker and rotavirus is reported in aggregate and

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by study arm (Tables 1 and S4). HumM2 and rotavirus were present in 0 to 9% of samples from

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the three environmental matrices. Mattioli et al.5 found similar rotavirus prevalence (3-9% of

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samples tested) in hand rinses and stored water in Tanzania. A previous study in Bangladesh

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found a substantially higher prevalence of rotavirus in tubewell water (40%) than we found in

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stored drinking water (0.6%).40 The difference may be due to a number of factors including


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different detection limits; Ferguson et al.40 filtered 2-8 liters of tubewell water, while we

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processed 100 mL of stored water.

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BacR was prevalent in all sample matrices; 22% of water samples and more than 50% of soil and

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hand rinse samples were positive. The high prevalence of ruminant fecal contamination may be

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due to the use of cow dung for domestic fuel. Avian-GFD was present in 9% of water, 16% of

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hand rinse, and 33% of soil samples.

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Ruminant-associated and avian GFD markers were more frequently detected than human

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markers in the compounds (z-test, p

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