Environmental indicators of enteric infections in a rural area and urban slum of Vellore, India.

Transcription

1 Environmental indicators of enteric infections in a rural area and urban slum of Vellore, India. Stefan Collinet-Adler, MD Masters of Public Health Applied Learning Experience Fall 2010 Tufts University Mentors: Elena Naumova, PhD Tufts University Boston, Massachusetts Vinohar Balraj, MBBS, MD, MSc, FFPH Professor at Christian Medical College Vellore, India 1

2 Table of contents Page number I. Introduction and Background 3 II. Study goals 4 III. Study objectives 4 IV. Review of the literature 5 V. Fly biology 7 VI. Summary of fly counting and trapping methods 9 VII. Pilot field trials prior to departure 11 VIII. Material and Methods 12 IX. Results 16 Linear regressions 19 Logistic regressions 21 X. Discussion 23 A) Linear regression modeling 23 B) Logistic regression modeling 25 C) Observations on the variability of fly densities 26 D) Observations on fly counting and trapping methods 27 E) The place of fly mitigation in public health interventions designed to decrease infectious diarrhea 28 F) Limitations 33 G) Future directions 34 XI. Conclusion 34 Acknowledgements 35 References 36 2

3 I. Introduction and background Globally, 4 billion cases of diarrhea occur annually with 88% thought to be secondary to unsafe water, inadequate sanitation and hygiene. It is estimated that globally 2.4 billion people live without access to clean drinking water through a lack of adequate sanitary facilities and deficient hygiene practices [1]. Diarrheal illnesses are a nuisance for immunocompetent well nourished hosts in the developed world. However, in the developing world, they are a major cause of infantile morbidity and mortality, estimated to cause approximately 1.87 million or 19% of deaths per year of children under the age of 5 [2]. Children in resource poor settings are particularly at risk not only for increased incidence of infectious diarrhea, but also for increased acute and long-lasting morbidity. Psychomotor and developmental stunting has been found to be associated with diarrheal infection, especially in children under 1 year of age, and its effects are still measurable many years after specific infections, such as Cryptosporidium [3-8]. Malnutrition is both a contributing factor to and a result of diarrhea [8-10]. These studies emphasize the need to prevent exposure of children to enteric pathogens. Diarrhea has been shown to be the cause of death in almost 23% of Indian children under the age of 5 [11]. Vellore District, in the southern state of Tamil Nadu in India, has been fertile ground for studies on enteric infections. Tufts Medical Center (TMC) and Tufts University have a rich history of productive collaboration with Christian Medical College (CMC) in Vellore. These collaborative efforts have provided insights into the epidemiology and implications of enteric infections in resource poor settings and have lead to several publications and grants funded by the US National Institutes of Health (NIH), Centers for Disease Control (CDC) and Indian Council of Medical Research (ICMR). A retrospective study collecting information from four urban areas of Vellore, between 1995 and 2003, found that diarrhea was the most common etiology of infant deaths, accounting for 49/219 infant deaths (22.4%). When neonatal deaths were excluded, diarrhea remained the most common etiology, accounting for 43/100 (43%) deaths [12]. A birth cohort study to investigate correlates of protective immunity to childhood rotavirus infections (CRI) was conducted by our collaborators at CMC in an urban slum of Vellore. This provided a significant reservoir of data with very important results. Reviewing all the findings is beyond the scope of this Applied Learning Experience (ALE); however, a brief summary of basic epidemiology is in order. Gastrointestinal illnesses were found to be the second most common cause of infant morbidity causing a median of 3 episodes per child-year. Diarrhea accounted for 889 of 3370 (26.4%) hospitalizations over the first year of life, second only to acute respiratory infections (1593/3370 or 47.3%)[13]. The highest diarrheal burden was found to be in the first year of life with decreasing morbidity thereafter [14]. Transmission of infectious diarrhea is fecal oral with zoonotic and/or anthroponotic cycles depending on the pathogen. The fate and transport of these pathogens in the natural and built environment is only partially understood. Many environmental parameters/indicators have been individually shown to reduce viability and infectivity of these pathogens. Factors that appear to be associated with the epidemiology of enteric pathogens include rain events, temperature, time dependent die-off of pathogens, water 3

4 currents, exposition to UV light, soil characteristics, vegetation cover, proximity of infective feces to water supplies ultimately used for consumption, type of water disinfection used, predation and sequestration of pathogens by invertebrates, soil and water ammonia levels, biofilm, desiccation, seasonality, pathogen reservoir characteristics such as number and age of animals, the use of un- or under-treated wastewater for agricultural irrigation, and pathogen transport by filth flies and other flying insects [15]. As part of a collaborative study funded by the CDC and ICMR on the role of environmental parameters in transmission of enteric infections, investigators from TMC and CMC are studying human diarrheal episodes for one year in cohorts from an urban slum (n=160) and a rural area of Vellore (n=140) (Tufts IRB #9257). Cases and causes of diarrhea are being documented by the microbiologic laboratory at CMC. This is a unique opportunity to confront simultaneous infectious and environmental data to characterize important determinants in the transmission of enteric infections. Through an exhaustive review of the literature, a list of the major environmental parameters liable to influence the transmission of diarrheal pathogens has been compiled, including weather, water quality and supply, soil properties, human/animal interactions, and fly populations. We anticipate that enteric infection will be more frequent and clustered in households with compromised water and food supplies either at the source or at the point of use. Compromised water and food will be associated with ineffective segregation of human and animal fecal material from these supplies. Ineffective segregation of waste from products of consumptions will be characterized and predicted by detectable conditions, such as increased fly densities, that are investigated in this ALE. High fly densities will be a function of food and waste management. It is hoped that the findings of this study will provide a solid foundation for designing effective interventions to combat enteric infections in resource poor settings. II. Study Goals Enhance understanding of transmission of enteric infections in semi-urban and rural communities in Vellore, India. III. Study objectives The primary objective of this investigation was to provide quantitative data on fly densities in the sites included in the Indo-USA study Environmental indicators of enteric infections in vulnerable populations. Flies are known to carry enteric pathogens and it is thought that some food-borne transmission of enteric infections may be mediated by flies [16]. Fly densities (fly counts over a specified period of time) were established in a standardized fashion for each household in the study. Fly densities alongside information from questionnaires on hygiene, food handling habits, human/animal interactions as well as water and soil measures will be used as predictors against the study s main outcome, namely number and severity of diarrheal episodes for each study child. We hypothesize 4

5 that high fly densities in a household will correlate positively with poor waste management and increased diarrhea. Stool samples from the study children are being tested for the major pathogens that cause infectious diarrhea. Pathogen detection on the flies collected could be used to strengthen a possible link between flies and infectious diarrhea. For this reason, the collected flies will be stored for future pathogen detection. Delays in recruitment have resulted in a lag in collection and analysis of diarrheal samples. Although this precludes the intended final analysis at this time, it has permitted a more in-depth study of the predictor variable, fly densities. This ALE evaluated fly density measurement methods and results and investigated several possible determinants of fly activity in four communities in Vellore district. IV. Review of the literature Enteropathogens previously isolated from flies in field and laboratory experiments include Campylobacter spp.[17-19], Salmonella spp.[17, 18, 20], Shigella spp. [17, 18, 21-23], Vibrio cholerae[17, 23], Escherichia coli including enterohemorrhagic Escherichia coli[17, 18, 20, 21, 23-26], Bacillus cereus [21], Cryptosporidium spp.[16, 27-29], Giardia lamblia[16], and rotavirus [30]. The three proposed mechanisms of potential pathogen transmission from flies to their environment include mechanical transfer from the exoskeleton, regurgitation, and fecal deposits [16, 17, 22, 25, 27-29, 31]. Many pathogens can survive on flies for up to 10 days [8]. A longitudinal study of enteric diseases in a Thai village underlines the importance of flies as a potential source of enteric pathogens in rural areas. Grid counts were conducted monthly for 1 year in animal pens, concrete-encased bathrooms (suams), yards, and kitchens. Sticky ribbons were placed for 24 hours in kitchens and net collections were performed in animal pens, yards and suams. The collection of flies revealed primarily Musca domestica (96% in kitchens, 92% in animal pens, 87% in suams and yards) with pathogens including E coli (ETEC), Shigella spp., and Vibrio spp. detected in 69%, 38%, 35%, and 8% of fly pools in yards, animal pens, suams, and kitchens respectively. Although a direct cause and effect could not be ascertained and directionality of any association would be uncertain, there was an increase in the fly population and the incidence of human diarrhea during the hot dry season. Furthermore, ETEC was only isolated in May and June, when the incidence of ETEC infections was highest in the human population [23]. A study in the late forties gave indirect evidence of transmission of shigellosis by house flies. Nine towns in Hidalgo County, Texas with high rates of diarrhea and high fly activities were chosen for a crossover interventional study using DDT to investigate the effects of fly control on morbidity due to diarrhea and the isolation of Salmonella and Shigella in rectal swabs of children under the age of 10 years. Fly control, measured using scudder grills (see fly trapping and counting methods below), was associated with a decrease in Shigellosis and to a lesser extent Salmonellosis. Incidence of diarrhea morbidity and mortality also decreased in the sprayed towns. A reversal of these trends 5

6 occurred when towns crossed over to no DDT. Re-treatment using insecticides was performed every 6 weeks, but spot retreatments at shorter intervals were sometimes needed. Fly populations rebounded quickly after discontinuation of DDT. The authors concluded that insecticides alone were not sufficient to control fly populations other than temporarily and hypothesized that elimination or disruption of breeding sites might be less expensive and more effective [32]. Limitations of this study include an uneven crossover design with one intervention lasting 17 months and the second lasting a mere 6 months. Moreover, the second interventional period did not include seasons with naturally high fly densities. There were also ethnic and therefore probable cultural and possible urban development differences between the two groups of towns, with the towns receiving 17 months of DDT having a higher non-hispanic population. Since the Hispanic populations had a higher burden of disease, sampling was confined to this group. Diarrhea morbidity was ascertained by monthly visits, introducing the real possibility of recall bias. A similar study was performed a few years later in an area of Georgia with low to moderate diarrheal morbidity. Once again, diarrhea and cultured Shigella declined in the treated areas but not in the untreated areas. Prevalence rates for these two outcomes were very similar in both areas prior to and after the intervention. Notably, after 10 months of DDT, resistance developed prompting the change to dieldren and chlordane. These two insecticides no longer controlled fly populations after approximately 12 weeks. When fly control became ineffective, the morbidity of the treated area rose to meet and even exceed the levels of the untreated area. Cross-over was not deemed practical as insecticide-resistant flies were present at the untreated sites, either from fly migration or from the use of household and agricultural insecticides [33]. A crossover study in Israel involving two military camps several miles distant and 4 sets of year old recruits was performed to investigate the results of intensive fly control using baited traps on Shigellosis. Two training sessions were evaluated, May to mid-july and August to mid-october over a two year period. These training camps were known to have high rates of diarrheal illness, particularly laboratory-proven shigellosis during the summer training periods. Routine fly control measures (periodic spot spraying with permethrin and covering refuse) were augmented by baited fly traps around the latrines, field kitchens, and mess tents in the intervention groups. Serologies for shigella and enterotoxogenic E. coli (ETEC) were performed using enzyme-linked immunosorbent assays (ELISA) at the beginning and end of each 11 week study period. Clinic visits with fecal swabs for microbiologic analysis were performed for diarrhea defined as at least 3 loose stools in a 24 hour period. Finally, environmental cultures including of stool in latrines as well as of pooled samples of flies were performed. Cohorts were comparable with respect to age, length of service, residence, ethnicity, education, or history of shigellosis. Mean fly counts performed using resting counts in early afternoon were 64% lower (p=0.024) in the intervention arms. During three of the four sessions, the incidence of diarrhea was significantly lower in the intervention arms, with shigellosis decreased by 85% (p=0.015) overall in the intervention arms compared to non-intervention. Overall, intensive fly control reduced the acquisition of antibodies to Shigella and ETEC by 76% (p=0.024) and 57% (p=0.006) respectively. Despite some methodologic limitations, it 6

7 remains one of only a handful of studies to convincingly suggest an association between flies and infectious diarrhea [34]. Another persuasive study on the possible link between flies and diarrhea in children under 5 is the crossover interventional trial over three years in 6 villages and two control villages in Pakistan where insecticide spraying using deltamethrin during the fly season from March to June was compared to no spraying for the first two years and the effect of baited traps was compared to no baited traps for the third year [35]. Preliminary studies in the area had shown that the seasonal peak in incidence of diarrhea coincided with peaks in fly densities. These rural villages had livestock kept in living compounds with manure heaps throughout the villages, domestic waste dumps, and the absence of pit latrines, thereby giving rise to adequate fly breeding and feeding potential. The control villages without any intervention revealed three pronounced annual fly seasons from March to June. Insecticide application essentially eliminated the fly populations in the sprayed villages while ongoing high fly densities occurred in the unsprayed villages, suggesting the effectiveness of spraying on controlling the fly populations. However, high fly densities were seen in all three groups the third year when baited traps were used as a fly control measure in one village, suggesting that this modality may be ineffective for fly control in certain settings. Interestingly, incidence of diarrhea in children under 5 as reported by mothers was significantly lower when villages were sprayed as compared to when villages were not sprayed or had never been sprayed (controls). There was a positive association between incidence of diarrhea and fly densities during the fly season (March to June), but the carry-over effect of spraying on fly densities was not associated with diarrhea incidence during the naturally low fly-density periods of the year. The investigators concluded that effective fly control, through insecticide use for example, may significantly reduce childhood diarrhea incidence when fly densities are elevated. Moreover, it was felt that the effect of fly control in this setting could rival that of World Health Organization (WHO) recommended diarrhea interventions such as immunization (e.g., rotavirus/cholera). Major limitations of this study include possible misclassification of diarrhea through the manner of documentation (interviews of the caretakers), the lack of etiologic investigation of diarrheal episodes, and no microbiologic evaluation of the flies captured to strengthen a possible epidemiologic link between flies and infectious diarrhea [35]. V. Fly biology Muscidae family: This family includes the common house fly (Musca domestica), the eye fly (Musca sorbens), the face fly (Musca autumnalis), and the lesser house fly (Fannia cannicularis), all of whom are well known as annoying pests to man. Musca domestica has world-wide distribution and are usually the most common fly invading homes. They live from 2-4 weeks. Females lay eggs in feces, carrion, and decomposing food. Entirely degraded organic substrates with low moisture content are not suitable for egg development. Ambient temperatures above and below C 7

8 prolong the hatching cycle and decrease the hatch rate. Adults feed on feces, human food, decaying organic matter. They are very strong fliers [36]. Fannia cannicularis has world-wide distribution and spends time on excrement, vertebrates, and rest on walls during short breaks and at night. It can vie with Musca domestica as the predominant indoor fly pest. It is notable for jerkily changing direction, often in the middle of rooms, where it circles hanging fixtures, such as lamps. Their lifespan is 2-3 weeks with an egg to egg cycle of days. They breed year round in frostless winters. They are more common in cooler periods of the year. Contrary to Musca domestica, observers have noted that they do not readily alight on humans, tables, or food. They lay eggs in semi-liquid organic matter, such as feces or decaying foods. Enclosed deep pit latrines have been shown to be refuges, breeding places, and food sources for Fannia species. They are attracted by sweat, mucous, and various fruits and have been reported to be associated with intestinal myiasis [37]. Callidiphoridae family: Commonly known as blow flies, about the size of Musca domestica or slightly larger, often with a metallic blue or green tint. They are scavengers, lay eggs in excrement, carrion, open sores, and other decaying organic matter. Dysentery has been associated with high blow fly populations and certain species are implicated in myiasis [38]. This family includes the Calliphora (blue bottle) and Lucilia/Phaenicia (green bottle) genuses. The green bottle fly adults prefer carrion for breeding, but all manner of garbage and animal excrement can also serve as breeding material. They are chiefly outdoor flies, more common in farm areas, and congregate around outdoor marketplaces, garbage, damaged fruit, carrion, open wounds, human and animal excreta. During the day, they live and rest outdoors, but in certain climes will enter houses in the spring and fall where they tend to alight on food rather than walls or other objects. Longevity of species such as Phaenicia sericata is days with averages ranging from 35 days in summer and 91 days in autumn in South Africa [37]. Chrysomya megacephala, or oriental latrine fly, also in the Callidiphoridae family, breeds in feces and carrion and can cause myiasis as well as transport enteric pathogens [39]. Liquid feces in large accumulations or fresh carrion are more efficient targets for development of eggs than city garbage, old carrion, or solid human feces. Adults live up to 54 days at C and 75% relative humidity with longer life spans at lower humidity [37]. They have a greenish-blue box-like body and feed on decaying organic matter, excreta and flowers. Sarcophagidae family: Commonly known as flesh flies, they are blackish with gray thoracic stripes and a checkered abdomen. They breed in carrion, feces, and decaying matter, with some species laying eggs in open wounds. Larvae (maggots) eat other insect larvae and carrion. Adults eat decaying vegetable matter, excrement and can be found around compost piles and latrines. They live for 5-10 days as maggots, then 5-7 days as adults. They have been implicated in myiasis. 8

9 Some authorities place Callidiphoridae and Sarcophagidae into the same family, Metopiidae [38]. Dispersion characteristics: The small mass of the fly relative to wingspan allows it to be carried by convection currents and wind. Passive transport on trucks and other vehicles as well as boats and aircraft is well documented. The marking, release and recapture of flies to study behavior have shown that flies will bypass clean areas for far more distant unsanitary conditions. Conversely the migration of house flies from unsanitary to more sanitary surroundings in urban areas could translate a degree of random dispersion or attraction to other unknown stimuli. Fly dispersal is canalized by topography as well as olfactory and other stimuli. Indole, trimethylamine, and skatole are products of meat decomposition and attract Callidiphoridae and Sarcophagidae more than Muscidae. Flies will fly upwind towards attractive sites even into winds up to 10 mph. Temperature and altitude affect speed and rate of flight. Some blow flies have increased flight activity with high humidity and rains. Complex influences of barometric changes on fly activity, possibly due to atmospheric ionic content have been noted. Maximum recorded flight distances for Musca domestica range from 300 meters to 32 kilometers with the most rapid dispersal speed being 3.2 kilometers in 5 hours recorded in a farming area of Manitoba in 1956 [37]. VI. Summary of fly counting and trapping methods Fly counts are the actual number of flies caught on a sticky ribbon. Fly counts are then divided by the number of days and portions of days they are left in place. These numbers are called fly density or fly abundance. These last two terms are used interchangeably in this report. It is hoped that measured fly density, or fly abundance, faithfully reflects fly activity. Some controlled animal experimental studies use the term fly density to describe the number of flies in a given volume. As in other field studies, the volume cannot be accurately measured in this report and the term density is used without regard to a measured volume. Fly counting can be accomplished using many different methods. -Spot cards are 7.5 x 10 cm file cards that are attached to surfaces using adhesive tape and record fecal and regurgitation spots produced by various types of flies. They are commonly used for fly activity surveillance in agricultural settings, give a good indication of overall fly activity, but do not permit fly identification or pathogen detection on flies. Spot cards are less useful if they become wet. They are most useful in sheltered environments. -Fly grids (Scudder grills) entail the use of squares with slats placed on a flat surface and are based on the propensity of Muscidae flies to alight on edges. The number of flies on the slats is counted by an observer over a given time period. This method is limited by the need for an observer to be present and concentrated; species can only be identified through a quick glance, possibly leading to misclassification; flies may be counted more 9

10 than once; pathogen detection on the flies is not possible; and the counts are limited by the fact that fly activity may vary greatly depending on time of day of observation. -Resting counts are performed by an observer or observers watching a specific area and counting the number of flies seen in that area. The limitations are the same as for fly grids. -Sticky fly ribbons are passive traps that catch flies without insecticides, are non-toxic, and rely on flies landing on or flying into the sticky part of the trap. These traps allow identification of fly types and pathogen detection on flies, do not count individual flies more than once, and can be left out for extended periods of time (days) without the need for the continued presence of an observer. These ribbons lose their fly catching characteristics with direct sun, high temperatures, rain or other high humidity conditions and therefore are most adapted to indoor settings. Vertically placed ribbons were four times more effective than horizontally placed ribbons placed in the same location within barns [37]. -Insecticide containing baited traps: Strips with sugar and an insecticide such as nithiazine attract and kill flies that are collected at the bottom of the trap. These traps allow identification of fly type, pathogen detection, and can be placed over long periods of time (hours to days) without an observer. The nithiazine is inactivated by sunlight and therefore is intended for indoor use. The presence of an insecticide makes prolonged human contact with this type of trap undesirable. -Baited jug traps are various types of containers with baits and colors that attract flies, allow easy entry through holes into a container, but have designs and color schemes that decrease fly escape from the container. These traps actively attract flies, are useful for fly counting and fly control, do not need constant observation, and do not count individual flies more than once. However, pathogen detection on the flies captured is compromised by the unknown microbiologic character of the bait and in cases using liquid bait, maceration of captured flies precludes accurate identification of fly types. These traps are usually most adapted to outdoor settings. Specific baits may attract certain families of flies preferentially, e.g. animal flesh bait may preferentially attract Calliphoridae [31]. Baited traps catch more flies in sheltered areas than in open areas [37]. -Nets can also be used to capture live flies for fly type identification and pathogen detection. This method has limitations in assessment of fly activity in that the sweeping motion modifies fly behavior, some indoor areas may be too cramped for efficient use of nets, an observer must be present, and evaluation of fly abundance over long periods of time (many hours to days) taking into account natural variation of fly activity is impractical. 10

11 VII. Pilot field trials prior to departure Field trials prior to departure: In order to test and validate fly counting methods and pathogen detection on flies, several types of traps were tested in indoor and outdoor sites in Massachusetts prior to departure. The choice of traps and methodology were guided by previously published literature [40, 41] and expert opinion (Christopher J Geden and David B Taylor, both with the United States Department of Agriculture). Given the aforementioned advantages and disadvantages of each method, three fly counting methods were retained for field testing, spot cards, sticky ribbons, and baited jar traps. Outdoor trapping: Initial funding concerns led us to consider field expedient baited jug traps with sugary bait. Rinsed out 1 gallon milk jugs were modified with four to six 1 cm diameter holes cut into the top part of the jug. Black paint was applied to the superior portion of the jug to limit escape of captured flies attracted to the holes. In previously published literature, painting the tops of these types of traps had resulted in a six-fold increase in fly capture [42]. The choice of bait was guided by results from field trials published in peer-reviewed journals, easy availability, cost, and the need for bait without insecticide or other toxic ingredients. Fly activity on dairy farms has been found to be high in areas where molasses is mixed with grain [41]. Jug traps baited with 25% dilutions of farm-grade blackstrap molasses with a small amount (5 grams) of detergent (Alcanox) as a surfactant were found to be just as effective as commercially available proprietary blend baits (Farnum ) during certain periods of a study [41]. Interestingly, premium grade molasses fit for human consumption was found to attract virtually no flies. Proprionic acid is often added to livestock grade molasses to slow spoilage. It is thought that this additive may be responsible for some of the attractiveness of this type of molasses to flies [41]. A study comparing water, honey, jaggery (widely available in India), maple syrup, and molasses as bait revealed that molasses was significantly more effective in attracting flies than the other sugary baits [42]. As we were to find, field expedient traps are far less effective at catching flies than commercially sold baited jug traps [42]. In late July and early August 2010, 300 milliliters of molasses diluted to 25% with tap water was placed in field expedient jug traps as described above and placed at two urban locations at Tufts Medical Center/Floating Hospital for Children in downtown Boston, Massachusetts. One was placed outside at 1 meter 50 cm from the ground immediately next to a garbage collection area 30 meters from an entrance to the Floating Hospital for Children. This garbage collection area did not have a foul odor. The second was placed in a sheltered area close to an open parking area immediately next to a large garbage collection bin with a distinctively foul odor. This trap was also placed at approximately 1 meter 50 cm from the ground and was left in place for one week. Visual evaluation of the two areas revealed few flies of the Muscidae and Callidiphoridae families. When retrieved, the first trap had captured no flies and the second had thousands of maggots from an unknown type of insect in the bait but no adult flying insects were trapped. Many tiny gnats were flying in and around the trap, but were not trapped given their small size. 11

12 During the same time period, baited jug traps were being field tested at an outdoor site (compost heap with visible Muscidae and Callidiphoridae activity) at the Natick Organic Community Farm in Natick Massachusetts. The field-expedient baited traps were again disappointing, capturing all manner of insects but not those of interest to this study. Sticky fly ribbons placed next to the baited jug trap were more successful with several Muscidae and Callidiphoridae caught over a 7 day period. The yield was less high than for indoor sticky ribbons placed during the same time period on the same farm (see below), possibly due to rain and direct sunlight decreasing the capturing qualities of this modality. It was thought that fly activity in the study sites in India would be higher, possibly making these types of traps useful in that context; however, it was decided that commercially available baited traps with proven efficacy would be used in India rather than risk disappointing results with home-made traps and sticky ribbons. Fliesbegone traps were decided upon given their proven effectiveness [42], acceptable price ($14 per trap), and favorable spatial geometry making shipping to India easier. Indoor trapping: Sticky fly ribbons were placed 1 meter 50 cm from the ground in the kitchen of Natick Organic Community Farm in Natick, Massachusetts. It was found that dozens of Muscidae and Callidiphoridae flies could be caught per week and per ribbon in an environment with modest visualized fly activity. The yield for these ribbons was higher at indoor sites than for outdoor sites with the fly densities encountered at this rural site in Massachusetts. Three spot cards were placed at 1 meter 50 cm from the ground in a barn without animals and with low visualized fly activity at Natick Organic Community Farm. Over a week, only two spots consistent with fecal spots/regurgitation spots were seen on one card, the other cards were unblemished. Pathogen detection: Flies caught using fly ribbons at Natick Organic Community Farm were tested for E. coli and Cryptosporidium using PCR techniques and were found to be negative. A second group of flies caught in the same manner were spiked with E. coli and Cryptosporidium and resulted in positive PCRs for these pathogens using gel electrophoresis. These pilot tests support the prediction that standard PCR techniques can be used to identify pathogens on flies and that the glue from the sticky ribbon probably present on parts of the flies would not interfere with detection of enteric pathogens. VIII. Materials and methods The existing collaborative study between CMC and TMC is multi-faceted. Community layouts with relationship of recruited homes with water supplies, fecal fields, sewage disposal and street locations have already been mapped and geo-referenced using Geographic Information System (GIS) technology by collaborators at CMC. Metereorologic data including rainfall, temperature, 12

13 humidity and sunshine indices are collected on a daily basis by the regional meteorologic office. This information has been and will continue to be available to investigators from CMC and TMC. Water purity at the source and at the point of use is analyzed for each recruited household once during each season over one year. Water usage and storage practices as well as food storage and preparation practices are evaluated by questionnaires. Interactions between domestic animals and people within the cohort households are also assessed using standardized questionnaires. Soil properties are also to be characterized. Stool collection with microbiologic analysis is performed during diarrheal episodes by CMC and TMC investigators. Fly counting and trapping were performed as described below. Study sites. This study took place in two rural villages, Kattuputhur and A. Kattupudi in Kaniyambadi block (a rural development block with an approximate population of 106,000 residents in 82 villages) and two urban slum areas, Ramanaickanpalayam (RNP) and Kaspa with a combined population of approximately 33,000. All of these sites are in Vellore District in the southern Indian state of Tamil Nadu. More detailed characteristics of the study sites can be found in Table 1. Meteorologic conditions. Weather measurements were taken from a commercial website (wunderground.com). These measurements are from Chennai, approximately 120 kilometers from Vellore. Historically, Vellore has had very similar weather to Chennai. The trends in Chennai during the fly trapping period closely mirrored trends in conditions experienced during this study including changes in temperature, humidity and precipitation. Fly trapping at the rural sites occurred from October 13-20, The mean average daily temperature during this period was 86.5 F with a range of F. The mean average daily humidity was 70.4% with extremes of %. Precipitation was 0 inches every day except 0.12 inches on October 16 th. Mean average daily wind speed was 6 mph with a range of 4-8 mph. Fly trapping at the urban sites occurred from October 20-30, The mean average daily temperature during this period was 83.5 F with a range of F. The mean average daily humidity was 85.5% with extremes of %. Precipitation was 0 inches except on October 26 (0.12 inches), October 28 (0.63 inches), October 29 (0.87 inches), and October 30 (0.2 inches). Mean average daily wind speed was 4.4 mph with a range of 3-6 mph. 13

14 Trapping methods Placement: Between October 13 th and October 30 th, 2010, fly ribbons were placed in 234 households in 2 urban sites and 2 rural sites in Vellore district in the Indian state of Tamil Nadu for 2.92 to 11 days (median 4.71 days). All traps were placed by one author (SCA) with the assistance of one field worker at the rural sites and two field workers at the urban sites. The sticky ribbons used were non-toxic passive traps (Revenge Fly Catcher ) placed in or as close to food preparation areas as was feasible by house geometry and as was acceptable to households, many of whom live in cramped quarters. Date and time (to the nearest hour) of placement was recorded. In order to increase fly trapping surface, every attempt was made to place the ribbons away from walls, but in a minority of cases (11/234), families insisted on having the traps placed against the wall to decrease the spatial burden of the ribbons on their day to day activities. In all cases, ribbons were placed away from direct sunlight and were sheltered from rain. House geometry and in some cases family preferences dictated the height of traps. The ribbons were placed such that the bottoms of the 61 cm traps were between 1 and 2 meters off the ground, with the majority being between 1m70 and 2 meters off the ground so as to be out of reach for the younger members of the families. Collection: Collection occurred several days after placement with date and time (to the nearest hour) of retrieval recorded. All traps that were placed and not removed by the families were collected and analyzed by one author (SCA) with the assistance of three field workers (one for the rural sites and two for the urban sites). In 8 cases (3.6%), the traps (6 rural and 2 urban) were collected independently by the same field workers after demonstrated proficiency in identification of flies to the Family taxon level using morphologic criteria and proper techniques for fly removal and placement in sterile tubes. Two forceps were used to place the flies from each household ribbon into previously sterile 1.8 milliliter tubes. Several tubes were necessary for household ribbons with more than 30 flies. The forceps were thoroughly cleaned with 70% isopropyl alcohol and completely air dried between ribbons to decrease the likelihood of cross-contamination between household samples. The tubes were then placed 1-6 hours after collection in a freezer at -70 C for ulterior microbiologic testing. Identification of flies was performed using morphologic criteria to the Family taxon level [36, 38, 43]. Two further methods, spot cards and baited fly traps, were initially employed to measure fly abundance. Questionnaires were administered by field workers in each house where a sticky ribbon was placed. These questionnaires explored demographic and hygiene characteristics of the household. Thirteen characteristics were retained as potential explanatory variables for fly activity: - Open air waste disposal in dugout pits in and around the living compound. Fly biology predicts that access to decomposing detritus will attract flies and encourage fly reproduction in the immediate vicinity of the household. 14

15 - Waste disposal in designated garbage areas/bins. Although these are also open air waste disposal areas, they are spatially further removed from living quarters than the previous variable and hence would be expected to attract flies away from household food preparation areas. - Possession of animals. This variable is an additive score of the pigs, dogs, cattle, goats, cats, and poultry possessed by a household. It does not take into account the exact location of the animals or the presence of other people s animals in close proximity to the household of interest. Animals are prone to haphazard defecation providing food sources and reproductive sites for flies. - No animals owned by the household or in the immediate neighborhood. This is also an additive score of the absence of animals in and around a household. A higher score reflects a more absolute lack of animals possessed or in the neighborhood. This score is likely to be a more robust measure of animal absence than the previous variable is a measure of animal presence. This difference is due to an unforeseen deficiency in the data gathering that imperfectly recorded location of animals. - Highest educational level attained by a member of the household. Education level has been shown to have an association with diarrhea [44-47]. Information on the educational level of all family members in households was collected. The maximum attained educational level in a household was retained for analysis. - Household income. Although this is a fairly standard variable to explore infectious diarrhea, poverty, and hygiene, its discriminatory value was found to be weak between these study sites. Mean and median incomes were not significantly different between the four sites and were not explanatory of fly densities in preliminary exploratory data analysis. Site of defecation, a more complex reflection of socio-economic status, was instead retained. - Access to a private indoor latrine was used to reflect inherited or acquired privilege. - Recourse to outdoor fecal fields for defecation was used as a marker for a lower socio-economic standing. - Use of cow dung by the family. This is an additive score of household use of cow dung. Some make cow dung patties for personal use or sale as an energy source. Others use cow dung as a building material in the walls or the floor. Finally, some use cow dung as fertilizer. Cow dung can conceivably attract flies; however, dried feces are much less attractive to flies than fresh feces. - The four sites. As can be seen in table 1, the sites differ significantly by a number of attributes. The two rural sites are relatively similar to each other, while there are some differences between the two urban areas. Overall, the urban and rural sites are significantly different from each other. Some of the site specific differences are captured by the other variables, but there remain unexplained differences, hence the utility of the sites as explanatory variables. Kaspa was used as the reference category as preliminary exploratory data analysis revealed that its capacity to explain fly densities was weak. Its fly densities were intermediary between the high values of the rural sites and the low values of its sister urban site, RNP. Previously, we performed secondary data analysis using a previous cohort study in the semi-urban areas that revealed that hygiene scores could be a predictor for number of 15

16 episodes of diarrhea (unpublished data). Hygiene scores may have been a helpful explanatory variable for fly densities in this study, but was not included as the scores were not felt to be sufficiently discriminatory between study households. As an example, nails trimmed, food covered, washing fruits and vegetables, and washing hands after defecation all had responses that were identical in % of cases. This was not the case for the previous cohort study. It is possible that asking the same questions of the same communities year after year generates homogeneous responses through true behavior modification or response bias. Statistical analyses and figure creation. All statistical analyses were performed using SPSS (SPSS, Inc., 2009, Chicago, IL, Identification, justification, and limitations of selected tests are included in the relevant results and discussion sections. Figures 1 and 2 were created with SPSS version Figures 3 and 4 were created with Visual Understanding Environment Version 3.0 (VUE-Academic technology, Tufts University, ). IX. Results Demographic and hygiene data by location are detailed in table 1. The sites are significantly different for most measured data with the clearest distinction being drawn between the rural and the urban sites. Two unifying characteristics for all four sites are low incomes and fairly similar family sizes. The proportions of various house types are not significantly different between the four sites. Kutcha homes are made with mud and thatch. Pucca homes are made with brick, cement and steel. The latter are usually sturdier than the former. The interior layout of homes, however, was very different from one household to the next. Of the ribbons placed, 224/234 (95.7%) were retrieved and analyzed 2.92 to 11 days (median 4.71 days) after placement. Of the ribbons placed, 10/234 (4.3%) were discarded by the family after placement and prior to retrieval. Five reasons for premature disposal were given: there are no flies in our home, the children ripped it down, my husband took it down, I don t know what happened to it, and a rat took it away. Thirteen of 224 (5.8%) of ribbons could not be placed in food preparation or consumption areas due to family objections or unsuitable hanging sites. Two thousand one hundred and one (2,101) flies were captured and counted with 2,095 (99.7%) from the Muscidae family (Musca and Fannia genus), 4 (2 per 1000) from the Callidiphoridae family, and 2 (1 per 1000) from the Sarcophagidae family. All other arthropods, lizards (4 geckos), and mammals (one small bat) caught by the ribbons were discarded. Characteristics of fly densities by location are detailed in table 2. 16

17 Table 1: Demographic and hygiene data by location. Item A Kattupudi Kattuputhur Kaspa RNP P-value Type of site Rural Rural Urban Urban Education (mean/sd */ */ #/ #/ (ANOVA-BF) median/mode) 11/8 10/10 10/10 10/10 Median income (itq range) 3000 ( ( ( (2500- NS 5000) 5750) 5000) 4500) Median family size (itq 5 (4-6) 5 (4-7) 5 (4-6) 5 (4-6) NS range) Pucca house 25/36 (69.4%) 17/35 (48.6%) 57/86 (66.3%) 45/64 (70.3%) NS Mixed house 2/36 (5.6%) 10/35 (28.6%) 14/86 (16.3%) 11/64 (17.2%) NS Kutcha house 9/36 (25%) 8/35 (22.9%) 15/86 (17.4%) 8/64 (12.5%) NS Hindu 18/36 (50%) 32/35 (91.4%) 46/86 (53.5%) 2/64 (3.1%) (Chi-square) Christian 18/36 (50%) 3/35 (8.6%) 2/86 (2.3%) 0/64 (0%) (Chi-square) Muslim 0/36 (0%) 0/36 (0%) 38/86 (44.2%) 62/64 (96.9%) (Chi-square) Cooking fuel: firewood 21/36 (58.3%) 14/35 (40.0%) 15/86 (17.4%) 2/64 (3.1%) (Chi-square) Cooking fuel: kerosene 2/36 (5.6%) 2/35 (5.7%) 25/86 (29.1%) 22/64 (34.4%) (Chi-square) Cooking fuel: LNG 13/36 (36.1%) 19/35 (54.3%) 40/86 (46.5%) 40/64 (62.5%) 0.05 (Chi-square) Living space-mean/sd** 0.46/ / / / (Anova-BF) Lack of animals (mean 5.25/1.42* 4.83/1.48* 6.56/0.85# 6.85/0.44# (Anova-BF) score/sd) Animals (mean score/sd) 1.06/1.12* 1.2/1.16* 0.16/0.43# 0.08/0.27# (Anova-BF) Use cow dung (mean 1.22/0.72* 1.43/0.88* 0.13/0.48# 0/0# (Anova-BF) score/sd) Use designated trash 9/36 (25%) 17/35 (48.6%) 48/86 (55.8%) 57/65 (87.7%) (Chi-square) areas/bins Trash disposal in dugout pits in or around compound 11/36 (30.6%) 18/35 (51.4%) 0/86 (0%) 0/65 (0%) (Chi-square, assumptions violated) Defecates in a fecal field 25/36 (69.4%) 21/35 (60%) 0/86 (0%) 0/65 (0%) (Chi-square) Use an indoor latrine 11/36 (30.6%) 12/35 (34.3%) 83/86 (96.5%) 65/65 (100%) (Chi-square) * and #: Values sharing one of these symbols are not significantly different one from the other; BF = Bon Ferroni post-hoc test; SD= standard deviation; itq = interquartile; NS = non-significant; **Number of rooms/number of family members. Table 2: Fly densities (Flies/day) by location. Site All A Kattupudi Kattuputhur Kaspa RNP Mean (SD) 2.12 (5.24) 5.19 (8.6) 3.80 (6.96) 1.63 (3.62) 0.16 (0.4) Median Mode Interquartile range Min-Max SD=Standard deviation The distributions of fly densities (fly abundances) throughout the entire sample and for two villages do not perfectly meet the assumptions of parametric testing (Figure 1) with departures from normality not entirely corrected by transformation (figure 2). Base 10 log transformation was used as it performed better than other attempted transformations. Using the non-parametric test independent samples Kruskal-Wallis, the distributions of fly abundance across the 4 sites were statistically different (p<0.001). Using the Mann Whitley U test, the distribution of fly abundances were -not different between A. Kattupudi and Kattuputhur (p=.703); 17

18 -significantly different between A. Kattupudi and RNP (p<0.001); -significantly different between A. Kattupudi and Kaspa (p=0.013); -significantly different between Kattuputhur and RNP (p<0.001); -significantly different between Kattuputhur and Kaspa (p=0.48); -significantly different between RNP and Kaspa (p<0.001). Figure 1 18

19 Figure 2 Study participants were asked by questionnaire whether flies were visible in their homes or not. Mean fly densities were found to be 0.17 (SD 0.35) for the 32 households stating that flies were not seen versus 2.52 (SD 5.67) for the 184 households reporting fly activity. The distribution of fly densities were statistically different between the two groups (<0.001 by Mann Whitney U test). Two further methods to measure fly abundance, spot cards and baited fly traps, were discontinued. Linear regressions Despite some violations of linearity, normality, and equality of variance, linear regressions were performed. The results for bivariate analysis can be found in table 3. A multivariable analysis can be found in table 4, while a multivariable linear regression after removal of problematic collinear predictors is shown in table 5. Table 6 is a more parsimonious multivariable linear regression. 19

20 Table 3: Bivariate analysis Log 10(fly abundances): Item B SE P-value Trash disposal in designated trash areas/bins Trash disposal in dugout pits in or around compound Possession of animals Absence of animals in home or neighborhood Household uses cow dung Highest educational level attained by a member of the household Family defecates in open fields/ drains Family defecates in indoor latrine Household income E-6 < Kattupudi Kattuputhur RNP Kaspa B=slope; SE= standard error. Table 4: Multivariable linear regression I using Log 10 (fly abundances): Item B SE P-value Constant Trash disposal in designated trash areas/bins Trash disposal in dugout pits in or around compound Possession of animals Absence of animals in home or neighborhood Household uses cow dung Highest educational level attained by a member of the household Household income 3.106E Family defecates in open fields/ drains Family defecates in indoor latrine Kattupudi Kattuputhur RNP B=slope; SE= standard error. R square Collinearity diagnostics suggested there might be redundancy involving latrine/open field defecation and animals/no animals. They could simply be describing the inverse one of the other and both terms are not needed. When outdoor defecation in a field and animal 20