• 1.

    WHO, 2009. Global Strategy for Infant and Young Child Feeding. Geneva, Switzerland: World Health Organization.

  • 2.

    Przyrembel H, 2012. Timing of introduction of complementary food: short-and long-term health consequences. Ann Nutr Metab 60: 820.

  • 3.

    Islam MS, Mahmud ZH, Gope PS, Zaman RU, Hossain Z, Islam MS, Mondal D, Sharker MAY, Islam K, Jahan H, 2013. Hygiene intervention reduces contamination of weaning food in Bangladesh. Trop Med Int Health 18: 250258.

    • Search Google Scholar
    • Export Citation
  • 4.

    Islam MA, Ahmed T, Faruque ASG, Rahman S, Das SK, Ahmed D, Fattori V, Clarke R, Endtz HP, Cravioto A, 2012. Microbiological quality of complementary foods and its association with diarrhoeal morbidity and nutritional status of Bangladeshi children. Eur J Clin Nutr 66: 12421246.

    • Search Google Scholar
    • Export Citation
  • 5.

    Henry FJ, Patwary Y, Huttly SRA, Aziz KMA, 1990. Bacterial contamination of weaning foods and drinking water in rural Bangladesh. Epidemiol Infect 104: 7985.

    • Search Google Scholar
    • Export Citation
  • 6.

    Kung’u JK, Boor KJ, Ame SM, Ali NS, Jackson AE, Stoltzfus RJ, 2009. Bacterial populations in complementary foods and drinking-water in households with children aged 10–15 months in Zanzibar, Tanzania. J Health Popul Nutr 27: 4152.

    • Search Google Scholar
    • Export Citation
  • 7.

    Afifi ZE, Nasser SS, Shalaby S, Atlam SA, 1998. Contamination of weaning foods: organisms, channels, and sequelae. J Trop Pediatr 44: 335337.

  • 8.

    Kotloff KL, Nataro JP, Blackwelder WC, Nasrin D, Farag TH, Panchalingam S, Wu Y, Sow SO, Sur D, Breiman RF, 2013. Burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the Global Enteric Multicenter Study, GEMS): a prospective, case-control study. Lancet 382: 209222.

    • Search Google Scholar
    • Export Citation
  • 9.

    Oo K, Sebastian A, Aye T, 1989. Carriage of enteric bacterial pathogens by house flies in Yangon, Myanmar. J Diarrhoeal Dis Res 7: 8184.

  • 10.

    De Jesüs AJ, Olsen AR, Bryce JR, Whiting RC, 2004. Quantitative contamination and transfer of Escherichia coli from foods by houseflies, Musca domestica L. (Diptera: Muscidae). Int J Food Microbiol 93: 259262.

    • Search Google Scholar
    • Export Citation
  • 11.

    Khalil K, Lindblom GB, Mazhar K, Kaijser B, 1994. Flies and water as reservoirs for bacterial enteropathogens in urban and rural areas in and around Lahore, Pakistan. Epidemiol Infect 113: 435444.

    • Search Google Scholar
    • Export Citation
  • 12.

    Butler JF, Garcia-Maruniak A, Meek F, Maruniak JE, 2010. Wild Florida house flies (Musca domestica) as carriers of pathogenic bacteria. Fla Entomol 93: 218223.

    • Search Google Scholar
    • Export Citation
  • 13.

    Echeverria P, Harrison BA, Tirapat C, McFarland A, 1983. Flies as a source of enteric pathogens in a rural village in Thailand. Appl Environ Microbiol 46: 3236.

    • Search Google Scholar
    • Export Citation
  • 14.

    Szalanski AL, Owens CB, McKay T, Steelman CD, 2004. Detection of Campylobacter and Escherichia coli O157: H7 from filth flies by polymerase chain reaction. Med Vet Entomol 18: 241246.

    • Search Google Scholar
    • Export Citation
  • 15.

    Nazni WA, Seleena B, Lee HL, Jeffery J, Rogayah TAR, Sofian MA, 2005. Bacteria fauna from the house fly, Musca domestica (L.). Trop Biomed 22: 225231.

    • Search Google Scholar
    • Export Citation
  • 16.

    Farag TH, Faruque AS, Wu Y, Das SK, Hossain A, Ahmed S, Ahmed D, Nasrin D, Kotloff KL, Panchilangam S, 2013. Housefly population density correlates with shigellosis among children in Mirzapur, Bangladesh: a time series analysis. PLoS Negl Trop Dis 7: e2280.

    • Search Google Scholar
    • Export Citation
  • 17.

    Collinet-Adler S, Babji S, Francis M, Kattula D, Premkumar PS, Sarkar R, Mohan VR, Ward H, Kang G, Balraj V, 2015. Environmental factors associated with high fly densities and diarrhea in Vellore, India. Appl Environ Microbiol 81: 60536058.

    • Search Google Scholar
    • Export Citation
  • 18.

    Graczyk TK, Knight R, Gilman RH, Cranfield MR, 2001. The role of non-biting flies in the epidemiology of human infectious diseases. Microbes Infect 3: 231235.

    • Search Google Scholar
    • Export Citation
  • 19.

    Kuhns DM, Anderson TG, 1944. A fly-borne bacillary dysentery epidemic in a large military organization. Am J Public Health Nations Health 34: 750755.

    • Search Google Scholar
    • Export Citation
  • 20.

    Irish S, Aiemjoy K, Torondel B, Abdelahi F, Ensink JHJ, 2013. Characteristics of latrines in central Tanzania and their relation to fly catches. PLoS One 8: e67951.

    • Search Google Scholar
    • Export Citation
  • 21.

    Meyer J, Shultz T, 1990. Stable fly and house fly breeding sites on dairies. Calif Agric 44: 2829.

  • 22.

    Nash J, 1909. House flies as carriers of disease. Epidemiol Infect 9: 141169.

  • 23.

    West LS, 1951. The House Fly its Natural History, Medical Importance, and Control. New York, NY: Comstock Publishing Company.

  • 24.

    Olsen AR, 1998. Regulatory action criteria for filth and other extraneous materials. III. Review of flies and foodborne enteric disease. Regul Toxicol Pharmacol 28: 199211.

    • Search Google Scholar
    • Export Citation
  • 25.

    Wolff H, Van Zijl W, 1969. Houseflies, the availability of water, and diarrhoeal diseases. Bull World Health Organ 41: 952959.

  • 26.

    Wright EP, 1983. The isolation of Campylobacter jejuni from flies. J Hyg (Lond) 91: 223226.

  • 27.

    Alam MJ, Zurek L, 2004. Association of Escherichia coli O157: H7 with houseflies on a cattle farm. Appl Environ Microbiol 70: 75787580.

  • 28.

    Shane SM, Montrose MS, Harrington KS, 1985. Transmission of Campylobacter jejuni by the housefly (Musca domestica). Avian Dis 29: 384391.

  • 29.

    Kobayashi M, Sasaki T, Agui N, 2002. Possible food contamination with the excreta of housefly with enterohemorrhagic Escherichia coli O157: H7. Jap J Sanit Zool 53: 8387.

    • Search Google Scholar
    • Export Citation
  • 30.

    Kobayashi M, Sasaki T, Saito N, Tamura K, Suzuki K, Watanabe H, Agui N, 1999. Houseflies: not simple mechanical vectors of enterohemorrhagic Escherichia coli O157: H7. Am J Trop Med Hyg 61: 625629.

    • Search Google Scholar
    • Export Citation
  • 31.

    Nayduch D, Pittman Noblet G, Stutzenberger F, 2002. Vector potential of houseflies for the bacterium Aeromonas caviae. Med Vet Entomol 16: 193198.

    • Search Google Scholar
    • Export Citation
  • 32.

    Sasaki T, Kobayashi M, Agui N, 2000. Epidemiological potential of excretion and regurgitation by Musca domestica (Diptera: Muscidae) in the dissemination of Escherichia coli O157: H7 to food. J Med Entomol 37: 945949.

    • Search Google Scholar
    • Export Citation
  • 33.

    Cohen D, Green M, Block C, Slepon R, Ambar R, Wasserman SS, Levine MM, 1991. Reduction of transmission of shigellosis by control of houseflies (Musca domestica). Lancet 337: 993997.

    • Search Google Scholar
    • Export Citation
  • 34.

    Fotedar R, 2001. Vector potential of houseflies (Musca domestica) in the transmission of Vibrio cholerae in India. Acta Trop 78: 3134.

  • 35.

    Hald B, Skovgård H, Bang DD, Pedersen K, Dybdahl J, Jespersen JB, Madsen M, 2004. Flies and Campylobacter infection of broiler flocks. Emerg Infect Dis 10: 14901492.

    • Search Google Scholar
    • Export Citation
  • 36.

    Holt PS, Geden CJ, Moore RW, Gast RK, 2007. Isolation of Salmonella enterica serovar Enteritidis from houseflies (Musca domestica) found in rooms containing Salmonella serovar Enteritidis-challenged hens. Appl Environ Microbiol 73: 60306035.

    • Search Google Scholar
    • Export Citation
  • 37.

    Chavasse DC, Shier RP, Murphy OA, Huttly SRA, Cousens SN, Akhtar T, 1999. Impact of fly control on childhood diarrhoea in Pakistan: community-randomised trial. Lancet 353: 2225.

    • Search Google Scholar
    • Export Citation
  • 38.

    Fukushima H, Ito Y, Saito K, Tsubokura M, Otsuki K, 1979. Role of the fly in the transport of Yersinia enterocolitica. Appl Environ Microbiol 38: 10091010.

    • Search Google Scholar
    • Export Citation
  • 39.

    Lindeberg YL, Egedal K, Hossain ZZ, Phelps M, Tulsiani S, Farhana I, Begum A, Jensen PKM, 2017. Can E. coli fly? The role of flies as transmitters of Escherichia coli to food in an urban slum in Bangladesh. Trop Med Int Health 23: 29.

    • Search Google Scholar
    • Export Citation
  • 40.

    Arnold BF, Null C, Luby SP, Unicomb L, Stewart CP, Dewey KG, Ahmed T, Ashraf S, Christensen G, Clasen T, 2013. Cluster-randomised controlled trials of individual and combined water, sanitation, hygiene and nutritional interventions in rural Bangladesh and Kenya: the WASH benefits study design and rationale. BMJ Open 3: e003476.

    • Search Google Scholar
    • Export Citation
  • 41.

    Simon AK, Hollander GA, McMichael A, 2015. Evolution of the immune system in humans from infancy to old age. Proc Biol Sci 282: 20143085.

  • 42.

    Guerrant RL, DeBoer MD, Moore SR, Scharf RJ, Lima AA, 2013. The impoverished gut—a triple burden of diarrhoea, stunting and chronic disease. Nat Rev Gastroenterol Hepatol 10: 220229.

    • Search Google Scholar
    • Export Citation
  • 43.

    Mashreky SR, Rahman A, Chowdhury S, Giashuddin S, Svanström L, Linnan M, Shafinaz S, Uhaa I, Rahman F, 2008. Epidemiology of childhood burn: yield of largest community based injury survey in Bangladesh. Burns 34: 856862.

    • Search Google Scholar
    • Export Citation
  • 44.

    Senior-White R, Aubertin D, Smart J, 1940. The Fauna of British India, Including the Remainder of the Oriental Region: Diptera. Family Calliphoridae, Vol. 6. New Delhi, India: Today & Tomorrow’s Printers & Publisher.

  • 45.

    Van Emden FI, 1965. The fauna of India and the adjacent countries. Diptera 7: 493541.

  • 46.

    Nandi BC, 2002. The Fauna of India and the Adjacent Countries: Diptera. Sarcophagidae, Vol. 10. Calcutta, India: Zoological Survey of India.

  • 47.

    Gilbert R, De Louvois J, Donovan T, Little C, Nye K, Ribeiro C, Richards J, Roberts D, Bolton F, 2000. Guidelines for the microbiological quality of some ready-to-eat foods sampled at the point of sale. PHLS Advisory Committee for Food and Dairy Products. Commun Dis Public Health 3: 163167.

    • Search Google Scholar
    • Export Citation
  • 48.

    Mickey RM, Greenland S, 1989. The impact of confounder selection criteria on effect estimation. Am J Epidemiol 129: 125137.

  • 49.

    Kusumaningrum H, Riboldi G, Hazeleger W, Beumer R, 2003. Survival of foodborne pathogens on stainless steel surfaces and cross-contamination to foods. Int J Food Microbiol 85: 227236.

    • Search Google Scholar
    • Export Citation
  • 50.

    Joosten H, Lardeau A, 2004. Enhanced microbiological safety of acidified infant formulas tested in vitro. South Afr J Clin Nutr 17: 8792.

  • 51.

    Isobe KO, Tarao M, Chiem NH, Minh LY, Takada H, 2004. Effect of environmental factors on the relationship between concentrations of coprostanol and fecal indicator bacteria in tropical (Mekong Delta) and temperate (Tokyo) freshwaters. Appl Environ Microbiol 70: 814821.

    • Search Google Scholar
    • Export Citation
  • 52.

    Rochelle-Newall E, Nguyen TMH, Le TPQ, Sengtaheuanghoung O, Ribolzi O, 2015. A short review of fecal indicator bacteria in tropical aquatic ecosystems: knowledge gaps and future directions. Front Microbiol 6: 308.

    • Search Google Scholar
    • Export Citation
  • 53.

    Lanata CF, 2003. Studies of food hygiene and diarrhoeal disease. Int J Environ Health Res 13 (Suppl 1): S175S183.

  • 54.

    Kusumaningrum HD, van Asselt ED, Beumer RR, Zwietering MH, 2004. A quantitative analysis of cross-contamination of Salmonella and Campylobacter spp. via domestic kitchen surfaces. J Food Prot 67: 18921903.

    • Search Google Scholar
    • Export Citation
  • 55.

    Gorman R, Bloomfield S, Adley CC, 2002. A study of cross-contamination of food-borne pathogens in the domestic kitchen in the Republic of Ireland. Int J Food Microbiol 76: 143150.

    • Search Google Scholar
    • Export Citation
  • 56.

    Curtis V, Cairncross S, Yonli R, 2000. Domestic hygiene and diarrhoea—pinpointing the problem. Trop Med Int Health 5: 2232.

  • 57.

    Pickering AJ, Julian TR, Marks SJ, Mattioli MC, Boehm AB, Schwab KJ, Davis J, 2012. Fecal contamination and diarrheal pathogens on surfaces and in soils among Tanzanian households with and without improved sanitation. Environ Sci Technol 46: 57365743.

    • Search Google Scholar
    • Export Citation
  • 58.

    Van Donsel DJ, Geldreich EE, Clarke NA, 1967. Seasonal variations in survival of indicator bacteria in soil and their contribution to storm-water pollution. Appl Microbiol 15: 13621370.

    • Search Google Scholar
    • Export Citation
  • 59.

    Mattick K, Durham K, Domingue G, Jorgensen F, Sen M, Schaffner DW, Humphrey T, 2003. The survival of foodborne pathogens during domestic washing-up and subsequent transfer onto washing-up sponges, kitchen surfaces and food. Int J Food Microbiol 85: 213226.

    • Search Google Scholar
    • Export Citation
  • 60.

    Talukdar PK, Rahman M, Rahman M, Nabi A, Islam Z, Hoque MM, Endtz HP, Islam MA, 2013. Antimicrobial resistance, virulence factors and genetic diversity of Escherichia coli isolates from household water supply in Dhaka, Bangladesh. PLoS One 8: e61090.

    • Search Google Scholar
    • Export Citation
  • 61.

    Harris AR, Pickering AJ, Harris M, Doza S, Islam MS, Unicomb L, Luby S, Davis J, Boehm AB, 2016. Ruminants contribute fecal contamination to the urban household environment in Dhaka, Bangladesh. Environ Sci Technol 50: 46424649.

    • Search Google Scholar
    • Export Citation
  • 62.

    Lindsay DR, Stewart WH, Watt J, 1953. Effect of fly control on diarrheal disease in an area of moderate morbidity. Public Health Rep 68: 361367.

    • Search Google Scholar
    • Export Citation
  • 63.

    Rahman MJ, Nizame FA, Nuruzzaman M, Akand F, Islam MA, Parvez SM, Stewart CP, Unicomb L, Luby SP, Winch PJ, 2016. Toward a scalable and sustainable intervention for complementary food safety. Food Nutr Bull 37: 186201.

    • Search Google Scholar
    • Export Citation
  • 64.

    Touré O, Coulibaly S, Arby A, Maiga F, Cairncross S, 2011. Improving microbiological food safety in peri-urban Mali; an experimental study. Food Control 22: 15651572.

    • Search Google Scholar
    • Export Citation
  • 65.

    Parvez SM, Kwong L, Rahman MJ, Ercumen A, Pickering AJ, Ghosh PK, Rahman M, Das KK, Luby SP, Unicomb L, 2017. Escherichia coli contamination of child complementary foods and association with domestic hygiene in rural Bangladesh. Trop Med Int Health 22: 547557.

    • Search Google Scholar
    • Export Citation
  • 66.

    Halder AK, Tronchet C, Akhter S, Bhuiya A, Johnston R, Luby SP, 2010. Observed hand cleanliness and other measures of handwashing behavior in rural Bangladesh. BMC Public Health 10: 545.

    • Search Google Scholar
    • Export Citation
  • 67.

    Curtis VA, Danquah LO, Aunger RV, 2009. Planned, motivated and habitual hygiene behaviour: an eleven country review. Health Educ Res 24: 655673.

    • Search Google Scholar
    • Export Citation
  • 68.

    Freeman MC, Stocks ME, Cumming O, Jeandron A, Higgins J, Wolf J, Prüss-Ustün A, Bonjour S, Hunter PR, Fewtrell L, 2014. Systematic review: hygiene and health: systematic review of handwashing practices worldwide and update of health effects. Trop Med Int Health 19: 906916.

    • Search Google Scholar
    • Export Citation
  • 69.

    Ehiri JE, Azubuike MC, Ubbaonu CN, Anyanwu EC, Ibe KM, Ogbonna MO, 2001. Critical control points of complementary food preparation and handling in eastern Nigeria. Bull World Health Organ 79: 423433.

    • Search Google Scholar
    • Export Citation
  • 70.

    Gilman RH, Skillicorn P, 1985. Boiling of drinking-water: can a fuel-scarce community afford it? Bull World Health Organ 63: 157.

  • 71.

    Geden CJ, 2005. Methods for monitoring outdoor populations of house flies, Musca domestica L. (Diptera: Muscidae). J Vector Ecol 30: 244.

  • 72.

    Geden CJ, Szumlas DE, Walker TW, 2009. Evaluation of commercial and field-expedient baited traps for house flies, Musca domestica L. (Diptera: Muscidae). J Vector Ecol 34: 99103.

    • Search Google Scholar
    • Export Citation
 
 
 

 

 
 
 

 

 

 

 

 

 

Prevalence and Association of Escherichia coli and Diarrheagenic Escherichia coli in Stored Foods for Young Children and Flies Caught in the Same Households in Rural Bangladesh

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  • 1 International Center for Diarrheal Diseases Research, Bangladesh, Dhaka, Bangladesh;
  • | 2 Stanford University, Stanford, California;
  • | 3 University of California, Berkeley, Berkeley, California;
  • | 4 Department of Environmental Health Sciences, Rollins School of Public Health, Emory University, Atlanta, Georgia;
  • | 5 Johns Hopkins Bloomberg School of Public Health University, Baltimore, Maryland

Consumption of contaminated stored food can cause childhood diarrhea. Flies carry enteropathogens, although their contribution to food contamination remains unclear. We investigated the role of flies in contaminating stored food by collecting food and flies from the same households in rural Bangladesh. We selected 182 households with children ≤ 24 months old that had stored foods for later feeding at room temperature for ≥ 3 hours. We collected food samples and captured flies with fly tapes hung by the kitchen. We used the IDEXX Quanti-Tray System (Colilert-18 media; IDEXX Laboratories, Inc., Westbrook, ME) to enumerate Escherichia coli with the most probable number (MPN) method. Escherichia coli–positive IDEXX wells were analyzed by polymerase chain reaction for pathogenic E. coli genes (eae, ial, bfp, ipaH, st, lt, aat, aaiC, stx1, and stx2). Escherichia coli was detected in 61% (111/182) of food samples, with a mean of 1.1 log10 MPN/dry g. Fifteen samples (8%) contained pathogenic E. coli; seven (4%) had enteropathogenic E. coli (EPEC) genes (eae and/or bfp); and 10 (5%) had enteroaggregative E. coli genes (aat and/or aaiC). Of flies captured in 68 (37%) households, E. coli was detected in 41 (60%, mean 2.9 log10 MPN/fly), and one fly (1%) had an EPEC gene (eae). For paired fly-food samples, each log10 MPN E. coli increase in flies was associated with a 0.31 log10 MPN E. coli increase in stored food (95% confidence interval: 0.07, 0.55). In rural Bangladesh, flies possibly a likely route for fecal contamination of stored food. Controlling fly populations may reduce contamination of food stored for young children.

INTRODUCTION

To ensure adequate nutritional intake, liquid or semisolid foods are recommended for children after the age of 6 months to complement breastfeeding.1,2 In the context of Bangladesh, such foods can be dedicated foods prepared for the children, or it can be any regular food that is cooked for the family for the day.35 Young children’s foods commonly comprise suji, a traditional recipe containing rice/wheat powder, milk, sugar/molasses, and khichuri, a preparation of rice with lentils and vegetables, and also regular rice.3 The introduction of liquid or semisolid foods can also increase the risk of enteric pathogen transmission to children if these foods are contaminated.47 In rural Bangladesh, foods stored for multiple feeding events over the course of several hours were found to have a high microbial count and were associated with diarrhea among children < 24 months old.4

Diarrheagenic Escherichia coli, rotavirus, and Shigella spp. are major causes of child diarrhea in South Asia.8 Studies have reported flies as potential carriers of different enteric pathogens such as E. coli and Shigella spp.915 Shigellosis is endemic in rural Mirzapur, Bangladesh,8 and an earlier study found an association between housefly density in rural compounds of Mirzapur and shigellosis among toddlers and preschool children.16 A study in Vellore, India, also found an association between increased fly densities and diarrheal events among rural families and urban slum dwellers; the majority of episodes occurred in children < 5 years old, and pathogens including Salmonella spp., norovirus, rotavirus, and E. coli were detected in flies.17

Houseflies frequently contact excrement, especially when it is poorly contained.18,19 Female flies often deposit their eggs on decayed, fermenting material such as human or animal feces and can spread fecal organisms to surrounding environments and their inhabitants.9,18,20,21

Although stored food can be contaminated with diarrheagenic pathogens through various pathways, flies can play an important role in transmitting pathogens to food, as uncovered stored food may attract flies.4,6,7,2225

Earlier studies mostly explored the quantity and type of pathogens carried by flies yet did not compare that with food contamination.9,1315,26,27 A few studies conducted controlled laboratory experiments to provide evidence of flies as a mechanical vector, and some also underscored the possibility of flies not only carrying but also fostering pathogen multiplication.10,2832 Epidemiological studies also investigated the role of flies in diarrheal pathogen transmission and human infection.17,3336 A community-randomized trial in rural Pakistan implemented fly control and observed a reduction in self-reported diarrhea.37 Some field studies experimentally exposed sterile food samples to wild flies and detected diarrheal pathogens, but these results are not generalizable to our study setting.22,38 A recent study in urban slums of Bangladesh experimentally exposed cooked rice in the kitchen areas to flies and reported a five-fold (95% confidence intervals [CIs]: 2.5–8.7) increased odds of uncovered rice being contaminated with E. coli if flies landed on it, and in 50% of the samples where flies landed, the average E. coli count was > 0.6 × 103 colony-forming unit/fly landing.39 These results suggest that flies can transmit high levels of fecal contamination to exposed food, especially in high contamination settings such as slums or markets. However, the extent of the contribution of flies to food contamination in the natural setting of rural households and how domestic food hygiene practices and ambient conditions affect this transmission pathway remain unclear. Our aim was to investigate if the fly species prevalent in rural food preparation areas were correlated with fecal contamination in household stored food. In this study, we enumerated E. coli in flies captured near the food preparation area and in foods stored for young children from the same households in rural Bangladesh to assess the association between contamination detected in flies versus food. We also tested both food and flies for the presence of diarrheagenic E. coli genes.

MATERIALS AND METHODS

Study design, population, and site.

The cross-sectional study presented here was nested within a large-scale randomized controlled trial (Water, Sanitation and Hygiene [WASH] Benefits) located in rural central Bangladesh.40 The trial comprised six intervention arms and a double-sized control arm. This was a cluster randomized trial where clusters were geographically pair matched and the trial detail has been described elsewhere.40 In our study, we included a subset of households enrolled in the sanitation and control arms of the WASH Benefits trial. We enrolled households between August 2013 and March 2014.

Eligibility criteria.

We followed a predefined selection criterion, which included households with mothers who reported that they were not exclusively breastfeeding their children, i.e., these mothers were feeding semisolid or liquid food, dedicated food specially prepared for child, or regular household food to complement breastfeeding. A second inclusion criterion was that the households had food stored at room temperature for ≥ 3 hours for later feeding of the target child. We surveyed all the households enrolled in the sanitation and control arms of the larger trial and selected the households that met the above criteria. The 3-hour minimum storage time was chosen because previous work suggests that food-borne bacterial growth typically reaches high levels after 4 hours of storage.6 The age range of ≤ 24 months was chosen, as children at this age are more vulnerable to diarrhea because of their immature immune system41; exposure to diarrheal pathogens through stored food in this age group can lead to diarrhea and hinder their growth and development.42 These inclusion criteria allowed us to enroll 182 WASH Benefits study households—85 from the sanitation arm and 97 from the control arm.

Data collection.

All data and sample collection activities were completed within a single visit to the target household. At first, the field team asked the caregiver whether the households had any food for children ≤ 24 months old that had been stored at room temperature for ≥ 3 hours. On confirmation from the caregiver, they hung three 1.5-feet long strips of sticky fly tapes (Revenge Fly Traps; Roxide Inc., New Rochelle, NY) beside the food preparation area. The fly tapes did not include any attractants, only adhesives that retained the flies. In rural Bangladesh, village residents often process raw food in the open courtyard and use a covered yard space with or without walls for cooking.43 The flies usually do not venture near the kitchen stove because of the emitted heat and smoke. Therefore, to maximize fly capture, the field team hung the fly tape near the food preparation area and recorded the time. Then, they collected a sample of stored food, predominantly suji (a traditional recipe containing rice/wheat powder, milk, and sugar/molasses) or khichuri (rice prepared with lentils and vegetables); they sampled plain cooked rice prepared for the family if no dedicated food was present. The food sample was collected in a 50-mL sterile tube using a sterile spoon. During sample collection, the field staff asked if the sampled food had been reheated after preparation and touched the food storage container to determine whether it was warm or cold; food was categorized to be hot only if there was visible steam, indicating that the food was reheated. They also recorded the temperature and humidity of the food storage location with a digital thermometer (AcuRite 00325; Chaney Instrument Co., Lake Geneva, WI).The field team administered a structured questionnaire on household sanitation facilities and food hygiene practices. The field team also conducted spot checks within the household compound (cluster of adjacent households that share the same courtyard and were built within a boundary) to observe the type and cleanliness of the latrine(s), presence of animal or human feces and food waste within the courtyard, and food storage practices. The team visited four to five households each day, and at the end of all household surveys (3.3 hours on average after hanging the fly tape, standard deviation [SD] = 0.9 hours), they recorded the number of flies captured and determined captured fly species using a simple visual identification chart adapted from The Fauna of British India series.4446 Then, they collected the fly from the middle of the tape with the most flies using sterilized forceps to place it in a sterile Whirl-Pak bag® (Nasco Modesto, Salida, CA). Food and fly samples were transported on ice to the International Center for Diarrheal Diseases Research, Bangladesh (icddr,b) field laboratory within 6 hours of collection for analysis.

In a subset of households where more than one fly was captured on the fly tapes (N = 49), a second fly was collected close to the center of the fly strip. The second fly was stored in buffered glycerol saline solution, transported to the Food Microbiology Laboratory at the icddr,b, and tested within 72 hours of collection for the presence of Shigella spp.

Laboratory sample processing.

Enumeration of E. coli.

Laboratory research assistants processed the food and fly samples and used the IDEXX Quanti-Tray System with Colilert-18 media for the detection and enumeration of E. coli with the most probable number (MPN) method. They thoroughly crushed the fly by applying pressure with a pestle from the outside of the bag to expose the alimentary tract. After adding 100 mL of sterile distilled water, they vigorously shook the bag and then diluted 1 mL of the fly sample solution with 99-mL sterile distilled water. For food sample testing, a 10-g aliquot was homogenized with 100 mL of distilled water for 1 minute using a sterile BagMixer bag and BagMixer® 400 CC® (Interscience Laboratory Inc., Woburn, MA) at speed 4 with gap at −3 mm, and 10 mL of the homogenized solution was diluted with 90-mL sterile distilled water. We pretested different dilutions for both food and fly samples to determine the ideal dilution factor to minimize samples with undetectable E. coli or E. coli exceeding the Quanti-Tray upper detection limit. With the selected dilution ratios, our minimum detection limits were 100 MPN/fly and 1 MPN/wet g food.

The laboratory staff also weighed a second 5-g aliquot of unhomogenized food and placed it in a drying oven overnight to determine the sample moisture content to calculate the dry weight and report bacterial concentration per dry gram of food.

Both food and fly samples were incubated at 44.5°C for 18–22 hours. To ensure quality control, we tested one field blank per sample collector per week, one laboratory blank per laboratory assistant per day, processed 10% field duplicates (two samples from one household), and 5% laboratory replicates (two aliquots from the same sample). One field blank was collected by each sample collector each week. While collecting regular samples, the sample collector also filled one Whirl-Pak bag with distilled water at the study household as a measure of the staff’s sterile technique. This blank was then tested in the laboratory for E. coli and fecal coliforms. If the field blank showed any growth, we considered that contamination had occurred during sample collection and reinforced aseptic precautions for sample collection. Approximately 1% of the tested blanks had positive growth, and we did not conduct any adjustment during data analysis because the percentage was low.

Detection of diarrheagenic E. coli and Shigella spp.

The research assistants at the field laboratory stored the E. coli–positive IDEXX Quanti-Trays in the refrigerator (4–8°C), and the samples were transported twice a month to the Food Microbiology Laboratory at icddr,b. After transportation, the E. coli isolates from positive wells from each Quanti-Tray were pooled and tested by multiplex polymerase chain reaction following the method used by Islam et al.4; to detect genes st, lt for enterotoxigenic E. coli (ETEC), eae, bfp for enteropathogenic E. coli (EPEC), aat, aaiC for enteroaggregative E. coli (EAEC), ial, ipaH for enteroinvasive E. coli (EIEC), and stx1, stx2 for Enterohemorrhagic E. coli (EHEC).4

The research assistants at the Food Microbiology Laboratory processed the additional flies collected to test for Shigella spp. They used a sterile micropestle to grind the flies and generate a suspension in 1 mL of buffer glycerol saline solution in a 1.5-mL Eppendorf tube. The suspension was then mixed with 9 mL of Shigella broth and incubated at 37°C for 18–24 hours for enrichment of Shigella spp. The enrichment broth was inoculated on three different culture media, including MacConkey agar, Hektoen enteric agar, and xylose lysine deoxycholate agar. Typical colonies from these plates were selected for further confirmation following the procedure described by Islam et al.4

Data analysis.

We calculated the proportion of stored food and fly samples positive for E. coli and diarrheagenic E. coli and defined highly contaminated food as having ≥ 100 MPN E. coli/dry g, consistent with previous studies.4,47 We log10 transformed E. coli MPN concentrations to estimate mean contamination levels and used a value of 0.5 to calculate the logarithm when no E. coli was detected.

To measure the association between exposure variables and food contamination, we estimated the change of the mean log10 MPN E. coli concentration using a generalized linear model. We considered the presence of unhygienic latrines as an exposure variable; a latrine was classified as unhygienic if satisfied any of the following conditions: 1) it did not have a pan with functional water seal, 2) had visible feces on the slab, or 3) drained into the nearby environment, such as a pond or ditch. Both fully uncovered and partially covered food samples were defined as uncovered stored food. We used a robust sandwich standard error estimator to account for village-level clustering while calculating 95% CIs.

We conducted bivariate analyses to explore factors associated with the E. coli concentration in stored foods. In the multivariate models, we retained variables that were significant at the 20% level in bivariate analyses.48 We created two multivariate models—one for the households where at least one fly was captured (N = 68) and the other to include all study households (N = 182). The second model excluded the concentration of E. coli in flies from the exposure variables so that we could analyze the relationship between food contamination and other exposures using data from all study households rather than just the subset where flies were caught.

Ethical considerations.

The field team obtained written informed consent from the caregiver of children ≤ 24 months old and the household heads. In case of caregivers who were < 18 years old, we obtained written informed consent from their parents. The study protocol was approved by the Ethical Review Committee at icddr,b.

RESULTS

Household characteristics.

The median age of the study children was 5.9 months (interquartile range [IQR] = 4.4–7.8), and 52% (95/182) were children aged < 6 months—the age group up to which the World Health Organization and the Government of Bangladesh recommend exclusive breastfeeding (Table 1). Mothers had a mean of 5.6 years of formal education (Table 1). More than half of the households (61%; n/N = 111/182) had a monthly income < USD 130 (Table 1).

Table 1

Household characteristics and stored food type and handling practices in rural households with children < 24 months old in rural Bangladesh

Sociodemographic status% (n/N)/(mean ± SD)/median (IQR)
Age of the children, median (IQR)5.9 (4.4–7.8)
Child aged 0–6 months52 (95/182)
Mother’s years of education (mean ± SD)5.6 ± 3.6
Household income < USD 13061 (111/182)
Study arm
 Sanitation*47 (85/182)
 Control53 (97/182)
Household sanitation status (spot check)
 Unhygienic latrine58 (99/172)
 Human feces present in courtyard4 (7/182)
 Animal feces present in courtyard87 (159/182)
 Food remnant/trash present in kitchen§14 (26/182)
 Recent episode of child diarrhea in the compound (self report)ǁ13 (24/182)
Food type
 Rice21 (39/182)
 Suji73 (132/182)
 Khichuri#6 (11/182)
 Food not reheated after preparation (spot check)90 (172/182)
Food temperature**
 Hot3 (5/182)
 Warm9 (16/182)
 Cold88 (161/182)
 Food container uncovered (spot check)††23 (41/182)
 Food cooled without lid (self report)30 (54/182)
 Food/dirt on serving plate (spot check)32 (56/173)
 Food/dirt on serving utensil (spot check)43 (45/105)
Food stored (self report)
 3–4 hours66 (120/182)
 > 4 hours34 (62/182)
 Storage time (median, IQR)4 (3–5)
 Temperature of food storage area °C (mean ± SD)28.8 ± 5.2
 % Humidity of food storage area (mean ± SD)72.5 ± 12
Fly status
 > 1 fly present37 (68/182)
 Fly density (fly/household), median (IQR)0 (0–1)

IQR = interquartile range; SD = standard deviation.

The sanitation intervention included sanitation mobilization and promotion, child potties, sani-scoop hoes to remove feces from household environments, and dual water-sealed pit latrine upgrades.

A latrine was classified as unhygienic if it did not have a pan with functional water seal, or have visible feces on the slab, or drain into the nearby environment, such as a pond or ditch.

We were unable to observe latrines in 10 households.

Remnant food particles from raw food processing or leftover food remnants.

ǁIf any of the child < 5 years old in the compound suffered from diarrhea within last 7 days.

A preparation of semolina with milk or water.

Rice prepared with pulse and vegetables.

The samples that had visible steam were classified as hot. To determine whether it was warm or cold, the data collector touched the food container.

Uncovered also included partially covered food samples.

At least one unhygienic latrine was present within 58% (99/172) of the study compounds, and the field team observed animal feces in most (87%) household courtyards (Table 1). The caregiver-reported 7-day diarrhea prevalence for children < 5 years old living in study compounds was 13% (Table 1).

Stored food storage practices.

Suji was the most common type of stored food available and was collected from 73% of study households (Table 1). The field team observed that 23% of sampled food was not completely covered, and 30% of caregivers reported cooling hot food without a lid (Table 1). Ninety percent of caregivers reported that the sampled food was not reheated after cooking, and the field team assessed that 88% of storage containers were cold. The median storage time of the collected samples was 4 hours (IQR = 3–5); the average food storage area temperature was 28.8°C (SD = 5.2), and the average humidity was 72.5% (SD = 12.0) (Table 1).

Food and fly samples with E. coli and diarrheagenic E. coli.

Of 182 stored food samples, we detected E. coli (≥ 1 MPN/dry g) in 111 (61%). Among these, the mean E. coli concentration was 1.1 log10 MPN/dry g; 16% of the food samples were highly contaminated (> 100 MPN/dry g). We also detected E. coli–specific pathogenic gene(s) in 8% of stored food samples. Enteropathogenic E. coli genes (eae and/or bfp) were isolated from 4% of samples and EAEC genes (aat and/or aaiC) from 5% (Table 2). None of the food samples had ETEC- (st, lt), EIEC- (ial, ipah), or EHEC-specific (stx1, stx2) genes.

Table 2

Proportion of stored foods and flies positive for Escherichia coli and diarrheagenic E. coli in rural households with children < 24 months old in rural Bangladesh

Food, N = 182 n (%)Paired samples, N = 68
Food n (%)Fly n (%)
E. coli ≥ 1 MPN/dry g food111 (61)44 (65)
E. coli ≥ 100 MPN/dry g food or fly30 (16)14 (21)41 (60)
E. coli log MPN, mean (95% CI)0.3 (0.1–0.5)0.5 (0.1–0.8)2.9 (2.6–3.2)
Diarrheagenic E. coli gene15 (8)5 (7)1 (1)
Enteropathogenic E. coli7 (4)2 (3)1 (1)
Enteroaggregative E. coli10 (5)3 (4)

CI = confidence interval; MPN = most probable number.

Of the 182 study households, at least one fly was captured in 68 (37%) households (Table 1). Musca domestica was the predominant species captured (94%, 241/256 of flies captured on the fly tape).

Of the 68 collected flies, 60% (41/68) were positive for E. coli, and the mean E. coli concentration was 2.9 log10 MPN E. coli/fly. One (1%) sample contained EPEC-specific pathogenic genes (eae and bfp), and no other type of diarrheagenic E. coli was found in flies (Table 2). None of the sampled flies (N = 49) grew Shigella spp. by culture.

Association between the presence of diarrheagenic E. coli and the concentration of E. coli in flies and food.

In bivariate analyses, the mean E. coli concentration in food increased by 0.35 log10 MPN for each log10 MPN increase in the mean E. coli concentration in flies (95% CI: 0.12, 0.58) and by 0.07 log10 MPN for each additional fly captured (95% CI: 0.02, 0.13). Storage area temperature was also associated with the level of food contamination (0.09 log10 MPN increase in E. coli for each 1°C increase in temperature; 95% CI: 0.06, 0.12). Suji had a mean E. coli concentration 0.69 log10 MPN lower than other stored foods (95% CI: −1.3, −0.11) (Table 3). There was no association between food contamination levels and having an unhygienic latrine or open feces observed within the compound (Table 3).

Table 3

Bivariate analyses of factors associated with the change of the Escherichia coli (log10 MPN E. coli/g) concentration in stored food samples

Factors affecting log10 MPN E. coli concentration in stored foods
Mean log10 MPN E. coli change (95% CI)P value
Log MPN E. coli in flies0.35 (0.12, 0.58)0.00
Fly density (fly/household)0.07 (0.02, 0.13)0.01
Unhygienic latrine−0.06 (−0.30, 0.18)0.62
Animal feces in the courtyard0.33 (−0.29, 0.94)0.30
Food remnant/trash in the kitchen0.03 (−0.44, 0.51)0.90
Recent episode of child diarrhea in the compound−0.14 (−0.71, 0.42)0.61
Food type (reference value is plain rice)
 Suji−0.69 (−1.3, −0.11)0.02
 Khichuri−0.13 (−1.2, 0.99)0.81
 Uncovered food (spot check)−0.03 (−0.52, 0.47)0.92
 Food cooled without lid0.34 (−0.08, 0.75)0.11
 Food reheated0.03 (−0.58, 0.65)0.92
 Warm stored food0.10 (−0.95, 1.2)0.85
 Cold stored food0.61 (−0.34, 1.6)0.21
 Food storage time0.02 (−0.05, 0.09)0.62
 Temperature of food storage area (°C)0.09 (0.06, 0.12)0.00
 Humidity of food storage area (%)−0.001 (−0.02, 0.02)0.89

CI = confidence interval; MPN = most probable number.

The estimations were generated using the generalized linear model.

Among the households where at least one fly was captured (N = 68), for each log10 MPN E. coli increase in flies, there was a 0.31 log10 MPN E. coli increase in stored food (95% CI: 0.07, 0.55), and for each 1°C increase in the mean storage area temperature, there was a 0.07 log10 MPN increase in food E. coli (95% CI: 0.01, 0.13) (Table 4). Stored food type, cooling food without lid, and fly density were not associated with mean food E. coli counts in multivariate analysis (Table 4). In the model that included all study households, we used the number of flies (fly/household) captured in the food preparation area instead of the E. coli concentration in flies. The mean food E. coli count increased by 0.05 log10 MPN for each additional fly captured (95% CI: 0.01, 0.10). For each 1°C increase in the mean food storage area temperature, there was a 0.07 log10 MPN increase in mean food E. coli levels (95% CI: 0.04, 0.10) (Table 4). However, cooling food without a lid and stored food type were not associated with increased log10 MPN E. coli in foods in the adjusted analysis.

Table 4

Final multivariate model presenting the factors associated with the change of concentration of Escherichia coli (log10 MPN E. coli/g) in stored food samples

Multivariate model for households with flies (N = 68)Multivariate model for all households (N = 182)
Mean log10 MPN E. coli change (95% CI)Mean log10 MPN E. coli change (95% CI)
Log10 MPN E. coli in flies0.31 (0.07, 0.55)Fly density (number of flies/household)0.05 (0.01, 0.10)
Temperature of food storage area (°C)0.07 (0.01, 0.13)Temperature of food storage area (°C)0.07 (0.04, 0.10)

CI = confidence interval; MPN = most probable number.

The estimations were generated using the generalized linear model.

On one occasion (1%), both stored food and fly samples from the same compound had EPEC-specific genes (eae) (Table 2). Seven percent of the stored food samples had one or more diarrheagenic E. coli gene in households where ≥ 1 fly was captured (Table 2).

DISCUSSION

Stored foods in rural Bangladeshi households were contaminated with diarrheagenic E. coli that could be consumed by children ≤ 24 months old. Both fly density and fly contamination levels were associated with food contamination levels, suggesting that flies may have a potential role in fecal contamination of foods stored for feeding of young infants. Particularly, houseflies were dominantly captured and frequently had a high E. coli count. Because houseflies are known to breed on open manure and feed on foods they get access to, they can be the potential link between fecal bacteria and contaminated foods.21,22,39

The current study did not find an association between the level of E. coli contamination in food and observed latrine cleanliness or presence of animal feces in the courtyard. This might be because flies are very mobile and may be acquiring fecal contamination from open feces or unhygienic latrines present in neighboring households. We did not track fly movements, but the existence of contaminated flies is indicative of the existence of contamination in the local environment. Moreover, contaminated flies in the neighborhood can pose a risk to all nearby households and their respective foods because flies are highly mobile.

Fly contamination in our study was associated with food contamination irrespective of whether food was covered during storage. Foods were stored with cover in most of our study households (77%), suggesting that flies had limited access. Yet, many of the stored food samples had E. coli, suggesting that food was exposed to contamination despite being covered. Flies typically move very fast and do not sit for long, and it is evident that flies can transfer high levels of fecal contamination within a few landings both in laboratory settings and field experiments.10,29,39 Moreover, flies commonly feed and defecate at the same time thus can transmit pathogens not only from their wings and legs but also from their gut.2932 In our study, flies were often carrying high numbers of E. coli and thus could potentially contaminate food even with brief direct contact with the food, or by contact with potential objects such as utensils, storing pots, or hands that are used to handle food.49

In addition to fly density and fly contamination levels, the current study identified an effect of temperature on food contamination levels, consistent with previous evidence. Temperature is a key factor facilitating bacterial growth in stored foods, and ambient temperatures in a tropical climate such as Bangladesh are ideal for rapid bacterial multiplication.4,5052 Storing food under refrigeration can reduce bacterial growth.50,53 However, refrigerators are expensive and require consistent electricity supply and are therefore not feasible or common in resource-scarce settings. We intended to determine whether flies have any role in contaminating foods stored for child feeding, and our results suggest, but do not prove, that flies may contribute to household stored food contamination in rural Bangladesh. Indeed, there are several limitations to our scientific inference.

First, we concurrently collected both food and fly samples and are unable to confirm that fly contamination preceded food contamination. It is also possible that flies obtained contamination from food contact instead of the reverse. However, given that flies frequently have contact with various sources of fecal contamination, it is unlikely that they were clean before they landed on the stored food and became contaminated solely because of their food exposure.

Second, it is possible that both the flies and food had a common source of contamination. To control for this, we measured a list of confounding variables in our study, such as unhygienic latrine, animal feces, and food trash. However, it is possible that flies picked up contamination primarily from sources we did not measure. For example, flies may have acquired fecal contamination from dishcloths in the kitchen area, and caregivers may have independently transported contamination from dishcloths in the kitchen area to food through contaminated hands.54 Flies can also pick up fecal bacteria from other potential reservoirs such as contaminated raw meat or fish, rotten food, leftover foods, wet surfaces in the kitchen area, and household soil—all of which have been previously reported to contain high numbers of E. coli and/or pathogens and are readily accessible to flies.49,5559

Third, it is possible that by testing the whole flies, the concentration of bacteria was higher than that transferred from their legs/vomit/feces. However, flies can transmit pathogens even with brief contact, which can multiply to substantial levels while the food is stored, even if the level of contamination, initially introduced into food is low.5,6,10,29,31,39,50

Finally, we did not track the movement of the flies and hence were unable to directly observe a fly accessing fecal contamination and transmitting that to the household food. However, the correlation between contamination of flies and stored foods indicates that flies were associated with the household food contamination, and further investigation, such as genetic fingerprinting or source tracking of isolates, is necessary to causally link food contamination to flies.60,61

Despite these limitations, there are multiple reasons to consider flies as risk factors for food contamination. Multiple previous studies have reported an association between fly density and diarrheal diseases.17,19,33,37,62 If fly control reduced diarrhea in other settings, food is a likely mediating factor that can lead to human infection, as flies, particularly houseflies, have a strong affinity toward food.18,2832,37,38 Our study findings align with the above evidence and underscore the possibility of flies contaminating foods in the natural setting of rural Bangladeshi households.

There are other factors that can enhance food safety; keeping the food covered and storing it in a cabinet may help to reduce the opportunity for fecal contamination, yet, it is difficult to keep the food covered while serving.6365 Flies may land on the food while serving, and whether the food is covered during storage, pathogens may multiply if the food is stored at room temperature.5,6,10,39,50,56 Hand washing at key points can reduce fecal transmission but requires consistent water supply near the latrine and the food preparation area, which is not always feasible in water scarce settings.6668 Reheating is uncommon because of the scarcity of cooking fuel, and inadequate reheating at lower temperature can actually enhance bacterial growth rather than limiting it.64,69,70 Our findings suggest that strategies to control flies might improve the microbiological quality of stored foods.

A potential area for future research could be to explore different fly control methods. For example, hygienic disposal of animal feces, which can include placing them in a covered hole (that also can be later used as fertilizers) outside the household compound, can be adopted to eliminate fly breeding sources from the courtyard.21 Flies can be also controlled using baited fly traps, fly tapes, and insecticide spray.37,71,72 However, different fly control interventions need to be tested for feasibility in the local context. In settings such as rural Bangladesh, future research on household food contamination should include feasible fly control interventions that may help to reduce contamination levels and will generate more definitive evidence.

Acknowledgments:

The icddr,b acknowledges with gratitude the study participants, the dedicated field team. The icddr,b is thankful to the governments of Bangladesh, Canada, Sweden, and the United Kingdom for providing core/unrestricted support.

REFERENCES

  • 1.

    WHO, 2009. Global Strategy for Infant and Young Child Feeding. Geneva, Switzerland: World Health Organization.

  • 2.

    Przyrembel H, 2012. Timing of introduction of complementary food: short-and long-term health consequences. Ann Nutr Metab 60: 820.

  • 3.

    Islam MS, Mahmud ZH, Gope PS, Zaman RU, Hossain Z, Islam MS, Mondal D, Sharker MAY, Islam K, Jahan H, 2013. Hygiene intervention reduces contamination of weaning food in Bangladesh. Trop Med Int Health 18: 250258.

    • Search Google Scholar
    • Export Citation
  • 4.

    Islam MA, Ahmed T, Faruque ASG, Rahman S, Das SK, Ahmed D, Fattori V, Clarke R, Endtz HP, Cravioto A, 2012. Microbiological quality of complementary foods and its association with diarrhoeal morbidity and nutritional status of Bangladeshi children. Eur J Clin Nutr 66: 12421246.

    • Search Google Scholar
    • Export Citation
  • 5.

    Henry FJ, Patwary Y, Huttly SRA, Aziz KMA, 1990. Bacterial contamination of weaning foods and drinking water in rural Bangladesh. Epidemiol Infect 104: 7985.

    • Search Google Scholar
    • Export Citation
  • 6.

    Kung’u JK, Boor KJ, Ame SM, Ali NS, Jackson AE, Stoltzfus RJ, 2009. Bacterial populations in complementary foods and drinking-water in households with children aged 10–15 months in Zanzibar, Tanzania. J Health Popul Nutr 27: 4152.

    • Search Google Scholar
    • Export Citation
  • 7.

    Afifi ZE, Nasser SS, Shalaby S, Atlam SA, 1998. Contamination of weaning foods: organisms, channels, and sequelae. J Trop Pediatr 44: 335337.

  • 8.

    Kotloff KL, Nataro JP, Blackwelder WC, Nasrin D, Farag TH, Panchalingam S, Wu Y, Sow SO, Sur D, Breiman RF, 2013. Burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the Global Enteric Multicenter Study, GEMS): a prospective, case-control study. Lancet 382: 209222.

    • Search Google Scholar
    • Export Citation
  • 9.

    Oo K, Sebastian A, Aye T, 1989. Carriage of enteric bacterial pathogens by house flies in Yangon, Myanmar. J Diarrhoeal Dis Res 7: 8184.

  • 10.

    De Jesüs AJ, Olsen AR, Bryce JR, Whiting RC, 2004. Quantitative contamination and transfer of Escherichia coli from foods by houseflies, Musca domestica L. (Diptera: Muscidae). Int J Food Microbiol 93: 259262.

    • Search Google Scholar
    • Export Citation
  • 11.

    Khalil K, Lindblom GB, Mazhar K, Kaijser B, 1994. Flies and water as reservoirs for bacterial enteropathogens in urban and rural areas in and around Lahore, Pakistan. Epidemiol Infect 113: 435444.

    • Search Google Scholar
    • Export Citation
  • 12.

    Butler JF, Garcia-Maruniak A, Meek F, Maruniak JE, 2010. Wild Florida house flies (Musca domestica) as carriers of pathogenic bacteria. Fla Entomol 93: 218223.

    • Search Google Scholar
    • Export Citation
  • 13.

    Echeverria P, Harrison BA, Tirapat C, McFarland A, 1983. Flies as a source of enteric pathogens in a rural village in Thailand. Appl Environ Microbiol 46: 3236.

    • Search Google Scholar
    • Export Citation
  • 14.

    Szalanski AL, Owens CB, McKay T, Steelman CD, 2004. Detection of Campylobacter and Escherichia coli O157: H7 from filth flies by polymerase chain reaction. Med Vet Entomol 18: 241246.

    • Search Google Scholar
    • Export Citation
  • 15.

    Nazni WA, Seleena B, Lee HL, Jeffery J, Rogayah TAR, Sofian MA, 2005. Bacteria fauna from the house fly, Musca domestica (L.). Trop Biomed 22: 225231.

    • Search Google Scholar
    • Export Citation
  • 16.

    Farag TH, Faruque AS, Wu Y, Das SK, Hossain A, Ahmed S, Ahmed D, Nasrin D, Kotloff KL, Panchilangam S, 2013. Housefly population density correlates with shigellosis among children in Mirzapur, Bangladesh: a time series analysis. PLoS Negl Trop Dis 7: e2280.

    • Search Google Scholar
    • Export Citation
  • 17.

    Collinet-Adler S, Babji S, Francis M, Kattula D, Premkumar PS, Sarkar R, Mohan VR, Ward H, Kang G, Balraj V, 2015. Environmental factors associated with high fly densities and diarrhea in Vellore, India. Appl Environ Microbiol 81: 60536058.

    • Search Google Scholar
    • Export Citation
  • 18.

    Graczyk TK, Knight R, Gilman RH, Cranfield MR, 2001. The role of non-biting flies in the epidemiology of human infectious diseases. Microbes Infect 3: 231235.

    • Search Google Scholar
    • Export Citation
  • 19.

    Kuhns DM, Anderson TG, 1944. A fly-borne bacillary dysentery epidemic in a large military organization. Am J Public Health Nations Health 34: 750755.

    • Search Google Scholar
    • Export Citation
  • 20.

    Irish S, Aiemjoy K, Torondel B, Abdelahi F, Ensink JHJ, 2013. Characteristics of latrines in central Tanzania and their relation to fly catches. PLoS One 8: e67951.

    • Search Google Scholar
    • Export Citation
  • 21.

    Meyer J, Shultz T, 1990. Stable fly and house fly breeding sites on dairies. Calif Agric 44: 2829.

  • 22.

    Nash J, 1909. House flies as carriers of disease. Epidemiol Infect 9: 141169.

  • 23.

    West LS, 1951. The House Fly its Natural History, Medical Importance, and Control. New York, NY: Comstock Publishing Company.

  • 24.

    Olsen AR, 1998. Regulatory action criteria for filth and other extraneous materials. III. Review of flies and foodborne enteric disease. Regul Toxicol Pharmacol 28: 199211.

    • Search Google Scholar
    • Export Citation
  • 25.

    Wolff H, Van Zijl W, 1969. Houseflies, the availability of water, and diarrhoeal diseases. Bull World Health Organ 41: 952959.

  • 26.

    Wright EP, 1983. The isolation of Campylobacter jejuni from flies. J Hyg (Lond) 91: 223226.

  • 27.

    Alam MJ, Zurek L, 2004. Association of Escherichia coli O157: H7 with houseflies on a cattle farm. Appl Environ Microbiol 70: 75787580.

  • 28.

    Shane SM, Montrose MS, Harrington KS, 1985. Transmission of Campylobacter jejuni by the housefly (Musca domestica). Avian Dis 29: 384391.

  • 29.

    Kobayashi M, Sasaki T, Agui N, 2002. Possible food contamination with the excreta of housefly with enterohemorrhagic Escherichia coli O157: H7. Jap J Sanit Zool 53: 8387.

    • Search Google Scholar
    • Export Citation
  • 30.

    Kobayashi M, Sasaki T, Saito N, Tamura K, Suzuki K, Watanabe H, Agui N, 1999. Houseflies: not simple mechanical vectors of enterohemorrhagic Escherichia coli O157: H7. Am J Trop Med Hyg 61: 625629.

    • Search Google Scholar
    • Export Citation
  • 31.

    Nayduch D, Pittman Noblet G, Stutzenberger F, 2002. Vector potential of houseflies for the bacterium Aeromonas caviae. Med Vet Entomol 16: 193198.

    • Search Google Scholar
    • Export Citation
  • 32.

    Sasaki T, Kobayashi M, Agui N, 2000. Epidemiological potential of excretion and regurgitation by Musca domestica (Diptera: Muscidae) in the dissemination of Escherichia coli O157: H7 to food. J Med Entomol 37: 945949.

    • Search Google Scholar
    • Export Citation
  • 33.

    Cohen D, Green M, Block C, Slepon R, Ambar R, Wasserman SS, Levine MM, 1991. Reduction of transmission of shigellosis by control of houseflies (Musca domestica). Lancet 337: 993997.

    • Search Google Scholar
    • Export Citation
  • 34.

    Fotedar R, 2001. Vector potential of houseflies (Musca domestica) in the transmission of Vibrio cholerae in India. Acta Trop 78: 3134.

  • 35.

    Hald B, Skovgård H, Bang DD, Pedersen K, Dybdahl J, Jespersen JB, Madsen M, 2004. Flies and Campylobacter infection of broiler flocks. Emerg Infect Dis 10: 14901492.

    • Search Google Scholar
    • Export Citation
  • 36.

    Holt PS, Geden CJ, Moore RW, Gast RK, 2007. Isolation of Salmonella enterica serovar Enteritidis from houseflies (Musca domestica) found in rooms containing Salmonella serovar Enteritidis-challenged hens. Appl Environ Microbiol 73: 60306035.

    • Search Google Scholar
    • Export Citation
  • 37.

    Chavasse DC, Shier RP, Murphy OA, Huttly SRA, Cousens SN, Akhtar T, 1999. Impact of fly control on childhood diarrhoea in Pakistan: community-randomised trial. Lancet 353: 2225.

    • Search Google Scholar
    • Export Citation
  • 38.

    Fukushima H, Ito Y, Saito K, Tsubokura M, Otsuki K, 1979. Role of the fly in the transport of Yersinia enterocolitica. Appl Environ Microbiol 38: 10091010.

    • Search Google Scholar
    • Export Citation
  • 39.

    Lindeberg YL, Egedal K, Hossain ZZ, Phelps M, Tulsiani S, Farhana I, Begum A, Jensen PKM, 2017. Can E. coli fly? The role of flies as transmitters of Escherichia coli to food in an urban slum in Bangladesh. Trop Med Int Health 23: 29.

    • Search Google Scholar
    • Export Citation
  • 40.

    Arnold BF, Null C, Luby SP, Unicomb L, Stewart CP, Dewey KG, Ahmed T, Ashraf S, Christensen G, Clasen T, 2013. Cluster-randomised controlled trials of individual and combined water, sanitation, hygiene and nutritional interventions in rural Bangladesh and Kenya: the WASH benefits study design and rationale. BMJ Open 3: e003476.

    • Search Google Scholar
    • Export Citation
  • 41.

    Simon AK, Hollander GA, McMichael A, 2015. Evolution of the immune system in humans from infancy to old age. Proc Biol Sci 282: 20143085.

  • 42.

    Guerrant RL, DeBoer MD, Moore SR, Scharf RJ, Lima AA, 2013. The impoverished gut—a triple burden of diarrhoea, stunting and chronic disease. Nat Rev Gastroenterol Hepatol 10: 220229.

    • Search Google Scholar
    • Export Citation
  • 43.

    Mashreky SR, Rahman A, Chowdhury S, Giashuddin S, Svanström L, Linnan M, Shafinaz S, Uhaa I, Rahman F, 2008. Epidemiology of childhood burn: yield of largest community based injury survey in Bangladesh. Burns 34: 856862.

    • Search Google Scholar
    • Export Citation
  • 44.

    Senior-White R, Aubertin D, Smart J, 1940. The Fauna of British India, Including the Remainder of the Oriental Region: Diptera. Family Calliphoridae, Vol. 6. New Delhi, India: Today & Tomorrow’s Printers & Publisher.

  • 45.

    Van Emden FI, 1965. The fauna of India and the adjacent countries. Diptera 7: 493541.

  • 46.

    Nandi BC, 2002. The Fauna of India and the Adjacent Countries: Diptera. Sarcophagidae, Vol. 10. Calcutta, India: Zoological Survey of India.

  • 47.

    Gilbert R, De Louvois J, Donovan T, Little C, Nye K, Ribeiro C, Richards J, Roberts D, Bolton F, 2000. Guidelines for the microbiological quality of some ready-to-eat foods sampled at the point of sale. PHLS Advisory Committee for Food and Dairy Products. Commun Dis Public Health 3: 163167.

    • Search Google Scholar
    • Export Citation
  • 48.

    Mickey RM, Greenland S, 1989. The impact of confounder selection criteria on effect estimation. Am J Epidemiol 129: 125137.

  • 49.

    Kusumaningrum H, Riboldi G, Hazeleger W, Beumer R, 2003. Survival of foodborne pathogens on stainless steel surfaces and cross-contamination to foods. Int J Food Microbiol 85: 227236.

    • Search Google Scholar
    • Export Citation
  • 50.

    Joosten H, Lardeau A, 2004. Enhanced microbiological safety of acidified infant formulas tested in vitro. South Afr J Clin Nutr 17: 8792.

  • 51.

    Isobe KO, Tarao M, Chiem NH, Minh LY, Takada H, 2004. Effect of environmental factors on the relationship between concentrations of coprostanol and fecal indicator bacteria in tropical (Mekong Delta) and temperate (Tokyo) freshwaters. Appl Environ Microbiol 70: 814821.

    • Search Google Scholar
    • Export Citation
  • 52.

    Rochelle-Newall E, Nguyen TMH, Le TPQ, Sengtaheuanghoung O, Ribolzi O, 2015. A short review of fecal indicator bacteria in tropical aquatic ecosystems: knowledge gaps and future directions. Front Microbiol 6: 308.

    • Search Google Scholar
    • Export Citation
  • 53.

    Lanata CF, 2003. Studies of food hygiene and diarrhoeal disease. Int J Environ Health Res 13 (Suppl 1): S175S183.

  • 54.

    Kusumaningrum HD, van Asselt ED, Beumer RR, Zwietering MH, 2004. A quantitative analysis of cross-contamination of Salmonella and Campylobacter spp. via domestic kitchen surfaces. J Food Prot 67: 18921903.

    • Search Google Scholar
    • Export Citation
  • 55.

    Gorman R, Bloomfield S, Adley CC, 2002. A study of cross-contamination of food-borne pathogens in the domestic kitchen in the Republic of Ireland. Int J Food Microbiol 76: 143150.

    • Search Google Scholar
    • Export Citation
  • 56.

    Curtis V, Cairncross S, Yonli R, 2000. Domestic hygiene and diarrhoea—pinpointing the problem. Trop Med Int Health 5: 2232.

  • 57.

    Pickering AJ, Julian TR, Marks SJ, Mattioli MC, Boehm AB, Schwab KJ, Davis J, 2012. Fecal contamination and diarrheal pathogens on surfaces and in soils among Tanzanian households with and without improved sanitation. Environ Sci Technol 46: 57365743.

    • Search Google Scholar
    • Export Citation
  • 58.

    Van Donsel DJ, Geldreich EE, Clarke NA, 1967. Seasonal variations in survival of indicator bacteria in soil and their contribution to storm-water pollution. Appl Microbiol 15: 13621370.

    • Search Google Scholar
    • Export Citation
  • 59.

    Mattick K, Durham K, Domingue G, Jorgensen F, Sen M, Schaffner DW, Humphrey T, 2003. The survival of foodborne pathogens during domestic washing-up and subsequent transfer onto washing-up sponges, kitchen surfaces and food. Int J Food Microbiol 85: 213226.

    • Search Google Scholar
    • Export Citation
  • 60.

    Talukdar PK, Rahman M, Rahman M, Nabi A, Islam Z, Hoque MM, Endtz HP, Islam MA, 2013. Antimicrobial resistance, virulence factors and genetic diversity of Escherichia coli isolates from household water supply in Dhaka, Bangladesh. PLoS One 8: e61090.

    • Search Google Scholar
    • Export Citation
  • 61.

    Harris AR, Pickering AJ, Harris M, Doza S, Islam MS, Unicomb L, Luby S, Davis J, Boehm AB, 2016. Ruminants contribute fecal contamination to the urban household environment in Dhaka, Bangladesh. Environ Sci Technol 50: 46424649.

    • Search Google Scholar
    • Export Citation
  • 62.

    Lindsay DR, Stewart WH, Watt J, 1953. Effect of fly control on diarrheal disease in an area of moderate morbidity. Public Health Rep 68: 361367.

    • Search Google Scholar
    • Export Citation
  • 63.

    Rahman MJ, Nizame FA, Nuruzzaman M, Akand F, Islam MA, Parvez SM, Stewart CP, Unicomb L, Luby SP, Winch PJ, 2016. Toward a scalable and sustainable intervention for complementary food safety. Food Nutr Bull 37: 186201.

    • Search Google Scholar
    • Export Citation
  • 64.

    Touré O, Coulibaly S, Arby A, Maiga F, Cairncross S, 2011. Improving microbiological food safety in peri-urban Mali; an experimental study. Food Control 22: 15651572.

    • Search Google Scholar
    • Export Citation
  • 65.

    Parvez SM, Kwong L, Rahman MJ, Ercumen A, Pickering AJ, Ghosh PK, Rahman M, Das KK, Luby SP, Unicomb L, 2017. Escherichia coli contamination of child complementary foods and association with domestic hygiene in rural Bangladesh. Trop Med Int Health 22: 547557.

    • Search Google Scholar
    • Export Citation
  • 66.

    Halder AK, Tronchet C, Akhter S, Bhuiya A, Johnston R, Luby SP, 2010. Observed hand cleanliness and other measures of handwashing behavior in rural Bangladesh. BMC Public Health 10: 545.

    • Search Google Scholar
    • Export Citation
  • 67.

    Curtis VA, Danquah LO, Aunger RV, 2009. Planned, motivated and habitual hygiene behaviour: an eleven country review. Health Educ Res 24: 655673.

    • Search Google Scholar
    • Export Citation
  • 68.

    Freeman MC, Stocks ME, Cumming O, Jeandron A, Higgins J, Wolf J, Prüss-Ustün A, Bonjour S, Hunter PR, Fewtrell L, 2014. Systematic review: hygiene and health: systematic review of handwashing practices worldwide and update of health effects. Trop Med Int Health 19: 906916.

    • Search Google Scholar
    • Export Citation
  • 69.

    Ehiri JE, Azubuike MC, Ubbaonu CN, Anyanwu EC, Ibe KM, Ogbonna MO, 2001. Critical control points of complementary food preparation and handling in eastern Nigeria. Bull World Health Organ 79: 423433.

    • Search Google Scholar
    • Export Citation
  • 70.

    Gilman RH, Skillicorn P, 1985. Boiling of drinking-water: can a fuel-scarce community afford it? Bull World Health Organ 63: 157.

  • 71.

    Geden CJ, 2005. Methods for monitoring outdoor populations of house flies, Musca domestica L. (Diptera: Muscidae). J Vector Ecol 30: 244.

  • 72.

    Geden CJ, Szumlas DE, Walker TW, 2009. Evaluation of commercial and field-expedient baited traps for house flies, Musca domestica L. (Diptera: Muscidae). J Vector Ecol 34: 99103.

    • Search Google Scholar
    • Export Citation

Author Notes

Address correspondence to Solaiman Doza, Environmental Interventions Unit, Enteric and Respiratory Infections Program, Infectious Diseases Division, 68, Shaheed Tajuddin Ahmed Sarani, Mohakhali, Dhaka 1212, Bangladesh. E-mail: solaiman@icddrb.org

Financial support: This research protocol/activity/study was funded by the Gates foundation, USA, grant number OPPGD759 and also received an additional funding from the World Bank. The icddr,b acknowledges with gratitude the commitment of the Gates foundation, USA, and the World Bank to their research efforts.

Authors’ addresses: Solaiman Doza, Musarrat Jabeen Rahman, Mohammad Aminul Islam, Leanne Unicomb, Sarker Masud Parvez, and Kishor Kumar Das, International Center for Diarrhoeal Disease Research, Bangladesh (icddr,b), Dhaka, Bangladesh, E-mails: solaiman@icddrb.org, rahman.musarrat@gmail.com, maislam@icddrb.org, leanne@icddrb.org, parvez@icddrb.org, and kdas@icddrb.org. Laura H. Kwong and Stephen P. Luby, Stanford Woods Institute for the Environment and Freeman Spogli Institute for International Studies, Stanford University, Stanford, CA, E-mails: lakwong@stanford.edu and sluby@stanford.edu. Ayse Ercumen, Division of Epidemiology, School of Public Health, University of California, Berkeley, Berkeley, CA, E-mail: aercumen@gmail.com. Amy J. Pickering, Department of Civil and Environmental Engineering, Tufts University, Medford, MA, E-mail: amy.pickering@tufts.edu. Abu Mohd Naser, Department of Environmental Health, Rollins School of Public Health, Emory University, Atlanta, GA, E-mail: abu.mohd.naser.titu@emory.edu. Sania Ashraf, Center for Global Health, Johns Hopkins Bloomberg School of Public Health University, Baltimore, MD, E-mail: saniashraf@gmail.com.

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