Hands and Water as Vectors of Diarrheal Pathogens in Bagamoyo

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Hands and Water as Vectors of Diarrheal Pathogens in Bagamoyo, Tanzania Mia Catharine Mattioli,† Amy J. Pickering,‡ Rebecca J. Gilsdorf,† Jennifer Davis,†,‡ and Alexandria B. Boehm†,* †

Environmental and Water Studies, Department of Civil and Environmental Engineering, Stanford University, Stanford, California 94305, United States ‡ Woods Institute for the Environment, Stanford University, Stanford, California 94305, United States S Supporting Information *

ABSTRACT: Diarrheal disease is a leading cause of under-five childhood mortality worldwide, with at least half of these deaths occurring in sub-Saharan Africa. Transmission of diarrheal pathogens occurs through several exposure routes including drinking water and hands, but the relative importance of each route is not well understood. Using molecular methods, this study examines the relative importance of different exposure routes by measuring enteric bacteria (pathogenic Escherichia coli) and viruses (rotavirus, enterovirus, adenovirus) in hand rinses, stored water, and source waters in Bagamoyo, Tanzania. Viruses were most frequently found on hands, suggesting that hands are important vectors for viral illness. The occurrence of E. coli virulence genes (ECVG) was equivalent across all sample types, indicating that both water and hands are important for bacterial pathogen transmission. Fecal indicator bacteria and turbidity were good predictors of ECVG, whereas turbidity and human-specific Bacteroidales were good predictors of viruses. ECVG were more likely found in unimproved water sources, but both ECVG and viral genes were detected in improved water sources. ECVG were more likely found in stored water of households with unimproved sanitation facilities. The results provide insights into the distribution of pathogens in Tanzanian households and offer evidence that hand-washing and improved water management practices could alleviate viral and bacterial diarrhea.

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INTRODUCTION Up to 1.2 million deaths are attributed to diarrheal diseases annually, with at least half occurring in sub-Saharan Africa.1−3 Infectious diarrhea is caused by a variety of human enteric pathogens that are transmitted via the fecal-oral route. Fecal material can be transmitted via several exposure pathways, including consumption of contaminated water or food, exposure to contaminated soil, interaction with fecal-contaminated vectors, as well as contact with fecal-contaminated hands.4 However, the relative contribution of different exposure pathways to the health burden of infectious diarrhea is not known. Drinking water supplies in low-income countries are often vulnerable to fecal contamination. Some households use “unimproved” drinking water sources, such as surface water or unprotected shallow wells, which are susceptible to contamination by open defecation, sewage, wastewater runoff, animal feces, and shallow groundwater contamination.5−7 Even households that use improved water sourcesparticularly those located at some distance from the home, such as public taps and wellsare regularly found to have stored drinking water that is contaminated with feces.7,8 During water fetching, transport, and subsequent storage, fecal material can be introduced through contact with contaminated containers, cups, and hands.9,10 In addition, per-capita water use in developing countries is typically much lower than industrialized © 2012 American Chemical Society

countries, with important implications for personal hygiene and the transmission of pathogens via contaminated hands.11 Interventions to reduce diarrhea can thus include investments that improve sanitation facilities and drinking water quality, increase water availability, promote hand washing with soap, and control vectors. Epidemiological research suggests that all of these efforts can reduce the incidence of diarrhea, but the literature suggests a wide range in the magnitude of observed effects. For example, interventions that promote hand washing with soap suggest that child diarrhea may be reduced by 31% to 47%.12,13 Improvement of sanitation facilities may reduce diarrhea morbidity by 7 to 57%.14 Fly control reduced diarrheal illness by 23% in Pakistan.15 Meta-analyses of pointof-use water treatment interventions identify health impacts ranging from no effect to a 42% reduction in child diarrheal illness.16,17 Installing an improved but shared water point, such as a borewell with hand pump, has been found to have negligible impacts on diarrhea morbidity, yet few rigorous studies exist.14 Given the variability in these outcomes, no clear consensus has emerged regarding which exposure pathway(s) account for the greatest share of the diarrheal disease burden. Received: Revised: Accepted: Published: 355

September 25, 2012 November 23, 2012 November 26, 2012 November 26, 2012 dx.doi.org/10.1021/es303878d | Environ. Sci. Technol. 2013, 47, 355−363

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Hand Rinse Samples. Prior to taking the hand rinse sample, the enumerator recorded the length of time since the female head of household had last washed her hands, and noted whether dirt was visible on her palms or underneath her fingernails. Following methods of Pickering et al., hand rinse samples were taken from the respondent in each participating household.10,26 This process involves the participant placing her hands, one at a time, into a 75 oz. sterile sample bag (VWR, Radnor, PA) containing 350 mL of sterile DI water. Field blanks of hand rinse sampling bags were run weekly to ensure sterility of hand rinsewater. Hand rinse samples were placed in a cooler on ice and transported to the laboratory for microbial analyses within 6 h of collection. Stored Water Samples and Water Management Practices. A sample of stored water that was intended for drinking and cooking was collected in each household. Enumerators inquired whether and how the water had been treated, and how long the water had been stored prior to sampling. Respondents were also asked to identify the water source from which the stored water had been collected. Enumerators documented whether or not the water storage container was covered and then asked each respondent to extract water in the manner she usually would, pouring it into a 55 oz. sterile sample bag (VWR, Radnor, PA). If chlorine was detected using a dip chlorine strip (Hach Co., Loveland, CO), sodium thiosulfate was added to the water sample bag immediately before collection to neutralize any chlorine present in the water. Samples were stored in a cooler on ice and transported to the laboratory for microbial analyses within 6 h of collection. Water Sources. The water source that each household respondent reported collecting her stored drinking water from was sampled. Samples were collected using aseptic technique with a 10% HCl-acid washed cube-container. Chlorine was neutralized with sodium thiosulfate as for stored water. Samples were stored on ice and transported to the laboratory for microbial analysis within 6 h of collection. Turbidity. The turbidity of the water and hand rinse samples was measured using a LaMotte 2020e/i Turbidity Meter (LaMotte Company, Chestertown, MD). See Supporting Information (SI) for more details. Fecal Indicator Bacteria Enumeration. E. coli and enterococci were enumerated using MI (BD Difco, Franklin Lakes, NJ) and mEI media (EMD, LaGrange, IL) following USEPA Methods 1604 and 1600, respectively.27,28 (See SI for details). Every 10th sample was run in duplicate. Approximately five method blanks were run per day. E. coli Pathogenic Genes. E. coli filters with E. coli biomass were removed from the MI media and placed in a 5 mL transport tube (E&K Scientific, Campbell, CA) containing 500 μL of RNAlater (Qiagen, Germantown, MD), and transported to Stanford University (Stanford, CA, USA) for molecular processing. Once at Stanford University, filters were stored at −20 °C until molecular analysis (6−12 months). Details of DNA extraction are in the SI. Three multiplex polymerase chain reactions (PCR) (Table S1 of the SI) were used to detect seven diarrheagenic E. coli virulence genes that are commonly found in Shigella spp., as well as five different pathotypes of E. coli including enteroinvasive E. coli (EIEC), enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC), enterotoxigenic E. coli (ETEC), and enterohemorragic E. coli (EHEC).29 The virulence genes tested were stx1 and stx2 (present in EHEC),

Indeed, the most important exposure pathway may vary by location depending on local circumstances. Another reason that it has proven difficult to characterize the relative importance of different exposure pathways for infectious diarrhea is that field measurements typically rely on detection of fecal indicator bacteria (FIB), rather than of actual enteric pathogens, to characterize health risks associated with exposure to water, soil, hands and surfaces. Testing for FIB (e.g., Escherichia coli and enterococci) requires fewer resources than testing for the many different pathogens that could cause diarrhea. At the same time, FIB have been shown to originate from both human and nonhuman fecal sources, and even from nonfecal sources,18−20 which calls into question their utility as a proxy for the risk of exposure to enteric pathogens. Moreover, the association between FIB in drinking water supplies and health outcomes such as diarrhea has proven difficult to demonstrate on a consistent basis.10,21 The present study contributes to the limited body of published research investigating the presence of enteric pathogens in water and on hands, and the relationship between pathogens, fecal indicator bacteria, and household water supply and sanitation service levels. We use data obtained from 93 households in Bagamoyo, Tanzania, each of which included an under-5 child. From each household we collected samples of household stored drinking water, the source water from which the stored drinking water was collected, and a hand rinse from the female head of household. We characterized each sample in terms of turbidity, FIB, and the presence of the human-specific Bacteroidales marker and enteric pathogens. Specific pathogens detected include enterovirus, adenovirus, rotavirus, and seven pathogenic E. coli virulence genes. These pathogens were chosen for analysis because rotavirus, pathogenic E. coli, and Shigella spp. are believed to be major viral and bacterial etiologies of childhood diarrhea,22,23 and enteroviruses and adenoviruses are also recognized as major etiological agents of gastroenteritis in children in the developing world.24,25 It should be noted, however, that protozoan pathogens may also be important but were not studied herein. We compare the presence of pathogens found in stored drinking water, in source water, and on hands; examine the relationship between microbial indicators and pathogen presence; and investigate the association between the presence of pathogens and the presence of improved water and sanitation.

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MATERIALS AND METHODS Study Site and Sample Frame. Ninety-three households in Bagamoyo, Tanzania (06°28′S, 38°55′E), each with at least one child under five years old, were enrolled in the study and visited during March 2010. Households, defined as groups of people that sleep and eat together in a dwelling on a regular basis, were selected using weighted random sampling from 15 villages. During each household visit, data were collected via personal interviews, and hand rinse and stored drinking water samples were taken. Household Interview Data. Interviews were conducted by local enumerators with the female head of household, and included questions about water consumption, household water and sanitation services, hand hygiene behavior, and household socioeconomic and demographic characteristics. Enumerators participated in a 4-week training, which included instruction on survey content, electronic data collection, sterile sampling technique, and involved extensive practice and pretesting in nonstudy enrolled households. 356

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eaeA (present in EHEC and EPEC), STIb and LTI (present in ETEC), ipaH (present in EIEC and Shigella spp.), and aggR (present in EAEC).30 Three hand samples, 9 stored water samples, and 4 source water samples positive for at least one of the seven target virulence genes were selected and sequenced with an ABI 3100 Genetic Analyzer (Protein and Nucleic Acid (PAN) Biotechnology Facility, Stanford, CA) to confirm that there was amplification of the intended targets (see SI). Filtration and Genomic DNA/RNA Methods for the Detection of Enteric Viruses and the Human Specific Bacteroidales Marker. Water and hand rinse samples were processed for the detection of enteric viruses and the human specific Bacteroidales marker by membrane filtration through 47 mm, 0.45 μm-pore size nitrocellulose filters (HA type filters, Millipore, Billerica, MA). Prior to filtering, 0.5 mL of 2.5 M MgCl2 was added to every 50 mL of water sample filtered to facilitate the capture of virus particles.31 The volumes filtered for hand rinse samples, stored water samples, and source water samples were 100 mL, 250 mL, and 2000 mL, respectively; at times the volume filtered was less due to differences in sample turbidity. Extraction blanks were run daily. Filters were treated with 500 μL of the RNA/DNA stabilizing agent RNAlater (Qiagen, Germantown, MD), allowed to sit for 5 min, and then vacuum aspirated.32 Filters were stored at −20 °C for 24 h and then placed at −80 °C for up to 5 months until transport to Stanford University for molecular analysis. Samples were transported to Stanford at room temperature. Once at Stanford, filters were stored at −80 °C until further molecular processing (storage of 6−12 months). Total RNA and DNA were extracted simultaneously from the filters using a modified MoBio PowerWater RNA Isolation Kit (Mo Bio Laboratories Inc., Carlsbad, CA) (see SI).29,33 Separate 25 μL aliquots of extracted nucleic acids were stored at −80 °C for subsequent molecular analyses. RNA/DNA aliquots underwent a maximum of one freeze−thaw cycle prior to molecular analysis. Virus and Bacteroidales assays. The three enteric viruses (enterovirus, rotavirus, and adenovirus) and the human-specific fecal marker Bacteroidales were detected using end-point PCR or RT-PCR with a hydrolysis probe on an Applied Biosystems StepOnePlus thermocycler (Applied Biosystems (ABI), Carlsbad, CA) (Table S1 of the SI). All samples were run in single reactions on 96-well plates (ABI, Carlsbad, CA). Standard plasmid DNA, cDNA, genomic RNA, or in vitro transcribed RNA was run with every end-point PCR and RT-PCR 96-well plate. Triplicate no template controls (NTC) were included in every run. Details of the molecular methods, adapted from published studies, are provided in the SI. The human-specific Bacteroidales marker was measured using the BacHum-UCD qPCR assay with the addition of Bovine Serum Albumin (BSA) (Invitrogen, Grand Island, NY);34 a method previously validated on human feces in East Africa.35 Enterovirus (EV) was detected using a modified version of the RT-QPCR protocol described in Walters et al.36 Human adenovirus (HAdV) was detected using a modified QPCR protocol from Jothikumar et al.,37 with the addition of Bovine Serum Albumin (BSA) (Invitrogen, Grand Island, NY). Human rotavirus was detected using a method RT-QPCR protocol from Jothikumar et al.38 The amplification threshold for the four assays was set to 0.50 ΔRn units, 0.02 ΔRn units, 0.20 ΔRn units, and 0.02 ΔRn units for the human Bacteroidales, enterovirus, adenovirus,

and rotavirus assays, respectively. A sample was considered positive if amplification occurred prior to a cycle threshold (Ct) of 44. The lowest detectable concentration of viruses and human-specific Bacteroidales for hand rinse samples, stored water samples, and source water samples (assuming amplification of 1 copy of target per reaction) was between 219 to 438 copies/2 hands, between 25 to 125 copies/100 mL, and between 3 to 125 copies/100 mL, respectively, depending on the volume of water filtered. To test inhibition, 28 hand samples, 26 stored water samples, and 20 source water samples were randomly selected and run in single reactions for adenovirus (DNA assay) and enterovirus (RNA assay). Each 25 μL PCR or RT-PCR reaction was spiked with 103 copies of the DNA or genomic RNA standard. The presence of inhibition was assessed by comparing Ct of the spiked sample with the Ct value of the 103 standard (referred to as ΔCt). Data Analysis. Data were analyzed using SAS Enterprise Guide version 4.3 (SAS Institute Inc., Cary, NC). FIB and turbidity were log10 transformed. If FIB were lower than the limit of detection, then half the detection limit was used. If FIB were too numerous to count (>500 CFU per filter), then the concentration of bacteria in the sample was calculated assuming 500 CFU per volume filtered. The variable “enteric virus” was defined as the presence/absence of at least one of the three enteric viruses. The variable “E. coli virulence gene” (ECVG) was defined as the presence/absence of at least one of the seven genes. Chi-square or Fisher’s exact tests (FET), as well as t tests, were used to analyze associations and differences of means and proportions between binary variables. Pearson correlation coefficients (rp) were used to evaluate the linear relationship between concentration measurements. Results were considered statistically significant at a significance level of p ≤ 0.01; some results are reported for 0.01 < p < 0.02.

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RESULTS Household Demographics. All of the respondents in the study were female heads of household aged 16 to 57 years. Average household size was 5.7 (SD 2.5, range 2−13) people, with an average of 1.2 (SD 0.5, range 1−4) children under the age of five per household. Of the 93 study households, 71 reported using an exclusive private latrine for their sanitation needs, 21 reported using a shared or neighbor’s latrine, and one reported practicing open defecation. Eighteen households reported using an “improved” sanitation facility according to the WHO/UNICEF Joint Monitoring Program 39 definition: Improved sanitation is defined as a pit latrine with a concrete slab; unimproved sanitation is defined as a pit latrine without a concrete slab, a shared latrine, or open defecation (complete definition is in the SI). Households reported collecting their stored drinking water from five types of sources, which were subsequently sampled: 74 collected their water from a municipal tap dispensing treated river water, 15 from a shallow well, 2 from a surface water source, 1 from a borewell, and 1 from harvested rainwater. Seventy-six (82%) households collected their water from “improved” sources according to the WHO/UNICEF Joint Monitoring Program39 definition: an improved water source is defined as a borewell, harvested rainwater, or a piped connection; an unimproved water source is defined as a shallow well or surface water (Complete definition is in SI). 357

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was detected only in hand rinses (not in the stored water or source water) in 4 households. Rotavirus was detected in the stored water and source water of 1 household. In 2 households, rotavirus was detected in the hand rinse and stored water. In 1 household, EV was detected in the source water and in the hand rinse. EV was detected in the hand rinse only in 9 households, and HAdv was detected in the hand rinse only in 5 households. The same virus was never found in all three types of samples within a single household. Within a household, there was a significant positive association between the presence of an enteric virus in a household’s stored water and the presence of an enteric virus in the household hand rinse sample (FET, p < 0.01). The same association was observed for rotavirus (FET, p < 0.02). No other statistically significant associations were observed. Pathogenic E. coli Virulence Genes. At least one of the seven pathogenic E. coli virulence genes (ECVG) measured was detected in 55 (59%) stored water samples, 42 (46%) source water samples, and 38 (41%) hand rinse samples (Table 1, Figure S1 of the SI). When results are examined in aggregate, the occurrence of the ECVG was not higher in any particular sample type. However, the individual virulence genes, eaeA (χ2= 6.4, p = 0.01) and stx1 (χ2 = 8.2, p < 0.01), were found significantly more often in household stored water than on hands. The detection of ECVG in hand rinses, stored water, and source water samples were compared within those households where measurements were available for all three sample types (n = 93). ECVG were detected in all three sample types within 14 households, while 16 households did not have the genes detected in any of the sample types. ECVG were detected in the hand rinse and stored water in 10 households, in stored water and source water in 15 households, and in the hand rinse and source water in 5 households. ECVG were detected only in the hand rinse (not in the stored or source water) in 9 households, only in stored water (not in the hand rinse or in the source water) in 16 households, and only in the source water of 8 households. Within a household, no associations were found between the presence of ECVG in the three sample types. The association of individual virulence genes in samples collected from the same household was also examined. The presence of ipaH (χ2 = 6.3, p = 0.01) and aggR (FET, p < 0.01) in a household’s stored water was significantly associated with its presence in the source water from which it was collected. The presence of the virulence gene aggR (FET, p = 0.01) in a household’s stored water was significantly associated with its presence in the hand rinse sample from the same household. No other statistically significant associations were observed. Human-Specific Bacteroidales. The human specific Bacteroidales marker (BacHum) was detected in 7 (8%) stored water samples, 4 (4%) source water samples, and 29 (32%) hand rinse samples. When data were examined in aggregate (Table 1), BacHum was equally likely to be found in household stored water and source water (χ2 = 0.9, p = 0.35). However, BacHum was more likely to be found on hands than in stored and source water (χ2 = 16.7, p < 0.01 and χ2 = 23.0, p < 0.01, respectively). The presence of BacHum within specific households for which all three sample types were analyzed (n = 86) was examined. BacHum was found only in a household’s stored water in 1 household, only in a household’s source water in 2 households, and only in hand rinses in 24 households. BacHum was found in both the stored water and the hand rinse sample

At the time of visit, respondents were storing an average of 6 (SD 4.3, range 1−22) 20-L buckets of water at their home. Chlorine was not detected in the stored water or in any of the source waters sampled. Even though chlorine was reportedly used at the nearby water treatment plant, it was never detected at the municipal taps. However, 15 (16%) households in the study reported treating their drinking water, some by two different methods: 12 by boiling, 4 filtering, 2 settling, and 1 chlorinating. At the time of interview, every household in the study had their stored water container covered. However, when collecting the stored water sample for microbiological analysis, ten respondents were observed to touch the stored water with their hands. On average, the households in our study reported using 9.1 (SD 4.7, range 3−20) liters of water per day per household for hand washing. Ten respondents had visible dirt on the palms of their hands, and 42 had visible dirt underneath their fingernails. The average length of time between hand rinse sampling and the respondent’s reported most recent hand washing with soap was 3.0 h (SD 2.5, range 0 - 16) (Table S2 of the SI). Enteric Viruses. Enterovirus (EV) was detected in 0 (0%) stored water samples, 1 (1%) source water sample, and 10 (11%) hand rinse samples. Rotavirus was detected in 3 (3%) stored water samples, 4 (4%) source water samples, and 8 (9%) hand rinse samples. Adenovirus (HAdv) was detected in 0 (0%) stored water samples, 0 (0%) source water samples, and 5 (6%) hand rinse samples (Table 1, Figure S1 of the SI). When Table 1. Percentage of Samples Positive for Pathogens Measured in the Three Different Sample Typesa

ECVG ipaH aggR LtIb STIb eaeA stx1 stx2 enteric virus rotavirus enterovirus adenovirus human Bacteroidales

hands

stored water

source water

N = 92/N = 90b

N = 93/N = 90b

N = 92

41.3 19.6 19.6 7.6 1.1 7.6 13.0 2.2 22.2 8.9 11.1 5.6 32.2

59.1 32.3 24.7 15.1 4.3 20.4 30.1 2.2 3.3 3.3 0.0 0.0 7.8

45.7 23.9 18.5 9.8 1.1 19.6 17.4 3.3 5.4 4.3 1.1 0.0 4.3

ECVG and ‘enteric virus’ indicate the presence of 1 or more E. coli virulence genes or enteric virus genes, respectively. bN = Number of viral and BacHum samples/N = Number of E. coli virulence gene (ECVG) samples. N < 93 in some cases because samples were lost. a

data were aggregated (Figure S1 of the SI, Table 1), the proportion of hand rinse samples with at least one enteric virus was greater than that of stored water samples (χ2 = 14.3, p < 0.01) or source water samples (χ2 = 10.4, p < 0.01). Enteric viruses were equally likely to be detected in stored water and source water samples (FET, p = 0.72). Comparisons for individual viruses were only possible for rotavirus (detection rates for EV and HAdv were prohibitively low), and the same trend was observed (FET, p < 0.01 for all comparisons). The detection of viruses among hand rinse, stored water, and source waters samples were compared within those households for which all three samples were analyzed (n = 86). Rotavirus 358

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Table 2. Comparison of the log-Mean of Fecal Indicator Bacteria (FIB) (E. coli and Enterococci) Concentrations when the Pathogen Is Detected (+) and Undetected (−) by Sample Type (T-test), and the Associations between the Presence of an Enteric Pathogen and the Presence of the Human Specific Bacteroidales Marker (N = No Association, Y = positive Association, χ2 Test)a,b,c hands

stored water d

N = 92/N = 90 E. coli ECVG ipaH eaeA aggR LtIb STIb stx1 stx2 enteric virus rotavirus adenovirus enterovirus human Bacteroidales

enterococci

+

−

+

−

2.82e 2.64 2.91 2.95e 2.64 4.48 2.60 2.32 2.61 2.63 2.58 2.89 2.63

2.12 2.34 2.36 2.26 2.38 2.37 2.37 2.40 2.33 2.37 2.38 2.33 2.28

3.08e 3.18e 3.18 2.85 3.17 2.54 3.20e 2.93 2.81 2.40 3.49e 2.93 2.82

2.28 2.47 2.56 2.55 2.56 2.61 2.52 2.60 2.55 2.63 2.56 2.57 2.50

source water d

N = 93/N = 90 E. coli

BacHum N N N N N N N N Ye N N N NA

enterococci

+

−

+

−

1.72e 1.80e 2.08e 1.67 1.82 2.13 1.69 2.19 2.36 2.36 − − 1.91

0.93 1.21 1.23 1.32 1.33 1.37 1.27 1.39 1.40 1.40 − − 1.39

1.87e 1.95e 1.90 1.89 1.83 2.01 1.88 2.29 2.30 2.29 − − 2.15

1.05 1.34 1.45 1.42 1.49 1.52 1.39 1.53 1.54 1.53 − − 1.51

N = 92 E. coli

BacHum N N N N N N N N N N − − NA

enterococci

+

−

+

−

1.80e 1.85e 2.24e 2.32e 1.72 3.70 2.42e 2.00 1.28 1.25 − 1.40 1.45

−0.02 0.50 0.47 0.48 0.72 0.78 0.48 0.78 0.79 0.80 − 0.81 0.79

1.77e 1.89e 2.12e 2.38e 1.67 3.70 2.23e 1.74 1.53 1.64 − 1.11 1.30

0.14 0.58 0.60 0.56 0.81 0.86 0.61 0.86 0.85 0.85 − 0.88 0.87

BacHum N N N N N N N N N N − N NA

a ECVG and “enteric virus” indicate the presence of 1 or more E. coli virulence genes or enteric virus genes, respectively. b−, Comparison was not possible due to lack of pathogen presence in sample type. cNA the analysis was not applicable. dN = Number of viral and BacHum samples/N = Number of E. coli virulence gene (ECVG) samples. N < 93 because some samples were lost. eStatistically significant (p ≤ 0.01).

Contamination in Improved Verses Unimproved Source Water and Sanitation. ECVG were more frequently detected in unimproved sources than in improved source waters (χ2 = 24.8, p < 0.01). At the individual gene level, the virulence genes ipaH, stx1, eaeA, and aggR were also more frequently found in unimproved source waters (p < 0.01 for all). Households that use unimproved water sources were more likely to have ECVG detected in their stored water as compared to households that use improved sources (χ2 = 7.3 p < 0.01); however, this difference was not significant when considering individual genes. No trends were observed for BacHum or enteric viruses (either the aggregate variable or the individual viruses). ECVG were significantly more frequently found in the stored water (χ2 = 6.2, p = 0.01) of households with unimproved versus improved sanitation facilities. At the individual gene level, stx1 was more frequently found in the stored water of households with unimproved sanitation facilities (χ2 = 6.4, p = 0.01). There were no associations observed between ECVG or any of the individual virulence genes in hand rinses and household sanitation. There were also no trends observed for BacHum or enteric viruses in either stored water or hand rinses. Turbidity. FIB levels and turbidity measurements were significantly and positively correlated with one another for all three sample types (Table 3, p < 0.01). Turbidity was significantly higher when at least one pathogenic E. coli virulence gene was present in stored water or in source water, but not in hand rinse samples (stored water: mean difference = 0.5 log units, t = 3.6, p < 0.01; source water: mean difference = 0.8 log units, t = 4.5, p < 0.01, hand rinse: mean difference = 0.2 log units, t = 2.2, p = 0.03). At the individual gene level, logmean turbidity measurements were significantly higher in stored water when the virulence genes eaeA or aggR were present and in source water when the virulence genes ipaH, eaeA, aggR, and stx1 were present (Table 3). Turbidity was significantly higher in hand rinse samples when an enteric virus

in 4 households and in both the stored water and the source water in 2 households. BacHum was never found in all three sample types for a given household. Within a household, there were no significant associations between the presence of BacHum in the different sample types. Enteric Pathogens and FIB. The concentrations of EC and ENT were significantly higher when ECVG were detected in hand rinses (EC: mean difference = 0.7 log units, t = 3.6, p < 0.01; ENT: mean difference = 0.8 log units, t = 3.8, p < 0.01), in stored water (EC: mean difference = 0.8 log units, t = 4.3, p < 0.01; ENT: mean difference =0.8 log units, t = 4.4, p < 0.01), and in source water (EC: mean difference = 1.8 log units, t = 8.9, p < 0.01; ENT: mean difference = 1.6 log units, t = 7.2, p < 0.01) (Table 2). At the individual gene level, average EC concentrations were significantly higher on hands when the virulence gene aggR was present, in stored water when the virulence genes eaeA or ipaH were present, and in source water when the virulence genes ipaH, eaeA, aggR, and stx1 were present (all p ≤ 0.01). Average ENT concentrations were significantly higher on hands when the virulence gene stx1 was present, in stored water when the virulence gene ipaH was present, and in source water when the virulence genes ipaH, eaeA, aggR, and stx1 were present (Table 2) (all p < 0.01). The average ENT concentration in hand rinses was significantly higher when HAdv was detected in the rinses (by 1 order of magnitude, t = 6.1, p < 0.01). No other significant differences in EC and ENT concentrations were observed with the presence/ absence of either individual enteric viruses or the aggregate variable. The concentrations of EC and ENT were not associated with the BacHum in any of the sample types. The presence of BacHum in hand rinse samples was associated with the presence of an enteric virus (χ2 = 9.1, p < 0.01) on hands, but not with occurrence of any individual virus or the occurrence of ECVG. 359

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on hands was also associated with their presence in stored water. This finding is consistent with the idea that contaminated hands can influence stored water quality, as has been suggested to occur with FIB.9,10 In contrast, ECVG were detected with roughly equal frequency in stored water and on hands, suggesting that both hands and water pathways may be important for exposure to these pathogens. These results help elucidate health risks for the 3.1 billion peoplefully 46% of the global populationwho rely on non-networked sources and in-home water storage for their drinking, cooking, and hygiene needs.41 Both virus and ECVG were found in source water samples, and the latter were significantly more likely to be detected in unimproved water sources, such as unprotected shallow wells and surface water. ECVG were also detected with greater frequency in stored water that was collected from unimproved sources further supporting the notion that sources classified as “improved” are safer for consumption. Other virulence genes and enteric viruses did not show similar associations, although temporal variability in water quality could make it challenging to observe associations between source and stored water quality.6,42,43 Viruses and ECVG were detected in some improved water sources (borewells and municipal taps). The presence of both types of pathogens in some improved sources could indicate that fecal contamination may be present in groundwater, penetrating into the piped water infrastructure, or that municipal water treatment was not sufficient. Indeed, chlorine, which was reportedly used at the nearby water treatment plant, was never detected at the municipal taps. These findings suggest that improving drinking water source infrastructure at both improved and unimproved water sources to prevent fecal contamination may result in health improvements. Commonly used indicators of fecal contamination exhibited different patterns of association with pathogens. Enterococci and E. coli correlated well with the presence of ECVG in all sample types, but generally did not correlate with occurrence of enteric viruses. The lack of association between human viruses and FIB in water has been previously documented;44−46 it may be caused by differences in the persistence of the two types of microorganisms in water47 or by extra-enteric, “naturalized” sources of enterococci and E. coli.18,48,49 By contrast, the presence of BacHum was associated with enteric virus presence on hands, which is consistent with previous research of fecalcontaminated marine beaches.50 These findings are not surprising because BacHum and human enteric viruses are both exclusively of human origin. Taken together, these results suggest that traditional FIB may be valid indicators for bacterial pathogens, whereas BacHum may be a better indicator for human viruses. Turbidity was positively and consistently associated with the presence of FIB in all three types of samples, and was positively associated with the presence of ECVG in water samples and enteric virus presence in hand rinses. Thus, the presence of dirt in water or on hands may be indicative of microbial contamination. Pickering et al.29 found enteric viruses, pathogenic E. coli, and BacHum genes in soils on household plots in this area of Tanzania, which would explain why turbidity (i.e., dirt) in water and on hands is related to pathogen presence. The source of pathogens in the soils is unknown, but it may be human excrement that is not properly contained in sanitation facilities. Associations between turbidity and microbial contamination in water and hand rinse samples have been

Table 3. Comparison of the Log-Mean of the Turbidity of a Sample When the Pathogen Is Detected (+) and Undetected (−) by Sample Type (t-test)a

ECVG ipaH eaeA aggR LtIb STIb stx1 stx2 enteric virus rotavirus adenovirus enterovirus human Bacteroidales

hands

stored water

N = 92/ N = 90d

N = 93/ N = 90d

source water N = 92

+

−

+

−

+

−

1.4 1.3 1.4 1.3 1.5 1.5 1.4 1.4 1.5c 1.4 1.5 1.6c 1.5c

1.2 1.3 1.3 1.3 1.2 1.3 1.2 1.3 1.2 1.2 1.2 1.2 1.2

0.9c 0.9 1.1c 1.0c 1.1 1.1 0.9 1.2 1.0 1.0 − − 0.7

0.4 0.6 0.5 0.6 0.6 0.6 0.6 0.7 0.7 0.7 − − 0.7

1.2c 1.4c 1.6c 1.7c 1.1 2.0 1.4c 1.1 1.0 1.2 − 0.5 1.1

0.5 0.7 0.6 0.6 0.8 0.8 0.7 0.8 0.8 0.8 − 0.8 0.8

ECVG and “enteric virus” indicate the presence of 1 or more E. coli virulence genes or enteric virus genes, respectively. b−, Comparison was not possible due to lack of pathogen presence in sample type. c Means are significantly different (p ≤ 0.01). dN = Number of viral and BacHum samples/N = Number of E. coli virulence gene (ECVG) samples. N < 93 in some cases because some samples were lost. a

was present (mean difference = 0.3 log units, t = 2.7, p < 0.01) or when enterovirus alone was present (mean difference = 0.4 log units, t = 2.7, p < 0.01). Turbidity was also significantly higher in hand rinse samples when BacHum was present (mean difference =0.3 log units, t = 3.2, p < 0.01). No other significant trends were observed. Blanks, Inhibition Tests, and Sequencing Confirmation. All field, extraction blanks, and NTCs were negative. Duplicate results agreed for PCR analyses and generally agreed for culture-based measurements. Average ΔCt for hand rinse samples, store water samples, source water samples, and extraction blanks run for inhibition in the adenovirus assay were 0.67, 0.60, 0.72, and 0.14 respectively. Average ΔCt for hand rinse samples, store water samples, source water samples, and extraction blanks run for inhibition in the enterovirus assay were −0.41, −0.38, −0.10, and −0.10, respectively. Because average ΔCt for adenovirus and enterovirus assays were less than 1, we concluded inhibition was not a problem. We obtained good and fair quality sequence reads for LTI, STIb, stx1, stx2, aggR, eaeA, and ipaH from one to four samples. The sequences aligned with their intended targets in GenBank with between 98 and 100% maximum identity values (Table S3 of the SI).

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DISCUSSION Whereas fecal indicator bacteria (FIB) concentrations have been previously reported for source water, stored water, and hand rinse samples in Tanzanian households,10,40 this study presents the first known evidence of enteric virus and E. coli virulence genes (ECVG) being detected simultaneously in hand and water samples among households. Enteric virus genes were more likely to be detected in hand rinses as opposed to stored water, suggesting that under some conditions hands may play an important role, perhaps more important than water, in the transmission of enteric viruses. The presence of enteric viruses 360

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Environmental Science & Technology observed by others,5,40,51 suggesting that turbidity may serve as a lower-cost alternative to FIB and pathogen testing. The E. coli virulence gene most often detected on hands and in both stored and source water was ipaH, suggesting the presence of either Shigella spp. or the E. coli pathotype, enteroinvasive E. coli (EIEC).30 Interestingly, Shigella spp. was the only enteropathogen found in stool samples of children in Ifakara, Tanzania (located 309 km from our study site) that was positively associated with cases of diarrhea.23 Other studies in Ifakara and Dar es Salaam (located 36 km from our study site), Tanzania, found enteropathogenic and enteroaggregative E. coli (EPEC and EAEC) to be the most commonly present pathotypes in the stool of children with diarrhea.52,53 The present study found ipaH, eaeA, and aggR (indicative of EIEC/ Shigella spp., EPEC and EAEC, respectively) more frequently in unimproved water sources compared to improved, and ipaH and aggR showed positive associations between source and stored water samples. Also, we found the presence of aggR in stored water to be significantly associated with its presence both in hand rinses and source water. These results suggest that postcollection contamination of stored water by hands, as well as contamination from the original source, may contribute to the exposure of children to pathogenic E. coli through water. Several important limitations of this study should be noted. The detection of virulence genes does not necessarily indicate the presence of infective pathogens. Future work that detects infectious pathogens is needed, although this work is difficult to implement in field settings. The ECVG were detected in DNA obtained from membrane filters containing biomass of E. coli and other coliforms. Therefore, we cannot rule out the possibility that the virulence genes were present in other coliform, and not in E. coli.54 Next, whereas associations between the presence of pathogens found in hand rinse and water samples were tested, the results of these analyses should be carefully considered in light of the differences between the units of measurement for each sample typeper two hands for hand rinse samples and per 100 mL filtered for water samplesas well as the differences in detection limits, which were dependent on the volume of water filtered and, in turn, on the turbidity of the sample. Previous research has shown that bacterial and viral contamination of hands and drinking water can vary significantly over time,6,55 yet our cross sectional study design does not allow us to consider this variability. Finally, the results presented here are from a single site in East Africa. Further work should be done to investigate the relative importance of hands as vectors of diarrheal pathogens as exposure routes. Microbe contamination may be different elsewhere.

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ACKNOWLEDGMENTS

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REFERENCES

This study was supported by the National Science Foundation (SES-0827384) and by the Stanford University Shah Research Fellowship. The authors acknowledge Angela Harris, Michael Harris, Debbie Lee, Emily Viau, and Maggie Montgomery for their support in the field and the laboratory. We acknowledge our collaborators Salim Abdulla, Mwifadhi Mrisho, and Omar Juma at the Ifakara Health Institute in Bagamoyo, Tanzania. This project would not have been possible without the Tanzanian lab and field teams and participating households.

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ASSOCIATED CONTENT

S Supporting Information *

Detailed information on methods, Tables S1−S3, and Figure S1. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 650 724 9128; fax: 650 723 7058; e-mail: aboehm@ stanford.edu. Notes

The authors declare no competing financial interest. 361

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