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A pilot study of behavioral, environmental, and occupational risk factors for chronic kidney disease of unknown etiology in Sri Lanka

A pilot study of behavioral, environmental, and occupational risk factors for chronic kidney... bioRxiv preprint first posted online Nov. 10, 2019; doi: http://dx.doi.org/10.1101/837393. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. 1 Title: A pilot study of behavioral, environmental, and occupational risk factors for chronic 2 kidney disease of unknown etiology in Sri Lanka 4 Short Title: Chronic kidney disease of unknown etiology (CKDu) case-control study in Sri 5 Lanka 1,2 3 3,4 5 6 7 Jake M. Pry ; Wendi Jackson ; Ruwini Rupasinghe ; Guneratne Lishanthe ; Zied Badurdeen , 6 7 3 4 8 Tilak Abeysekara ; Rohana Chandrajith ; Woutrina Smith , Saumya Wickramasinghe 10 Centre for Infectious Disease Research Zambia, Lusaka, Zambia 11 School of Medicine, Washington University, St. Louis, MO, U.S.A. 12 School of Veterinary Medicine, University of California, Davis, U.S.A 13 Faculty of Veterinary Medicine and Animal Science, University of Peradeniya, Sri Lanka 14 Renal Research Centre, District Hospital, Girandurukotte, Sri Lanka 15 Faculty of Medicine, University of Peradeniya, Sri Lanka 16 Faculty of Science, University of Peradeniya, Sri Lanka 18 Corresponding author: jpry@wustl.edu 19 Jake Michael Pry 20 Department of Infectious Disease 21 Washington University 22 660 S. Euclid Ave. St. Louis, MO 63110 23 Mobile. +260 96 3088750 | +1 936-661-6885 bioRxiv preprint first posted online Nov. 10, 2019; doi: http://dx.doi.org/10.1101/837393. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. 24 ABSTRACT 25 Chronic kidney disease of unknown etiology (CKDu) was first recognized in Sri Lanka in the early 26 1990s, and since then it has reached epidemic levels in the North Central Province of the country. 27 The prevalence of CKDu is reportedly highest among communities that engage in chena and paddy 28 farming, which is most often practiced in the dry zone including the North Central and East Central 29 Provinces of Sri Lanka. Previous studies have suggested varied hypotheses for the etiology of 30 CKDu; however, there is not yet a consensus on the primary risk factors, possibly due to disparate 31 study designs, sample populations, and methodologies. The goal of this pilot case-control study 32 was to evaluate the relationships between key demographic, cultural, and occupational variables 33 as risk factors for CKDu, with a primary interest in pesticide exposure both occupationally and 34 through its potential use as an ingredient in brewed kasippu alcohol. A total of 56 CKDu cases 35 and 54 control individuals were surveyed using a proctored, self-reported questionnaire. 36 Occupational pesticide exposure and alcohol consumption were not found to be significant risk 37 factors for CKDu. However, a statistically significant association with CKDu was observed with 38 chewing betel (OR: 6.11, 95% CI: 1.93, 19.35), age (OR: 1.07, 95% CI: 1.02, 1.13), owning a pet 39 dog (OR: 3.74, 95% CI: 1.38, 10.11), water treatment (OR: 3.68, 95% CI: 1.09, 12.43) and pests 40 in the house (OR: 5.81, 95% CI: 1.56, 21.60). The findings of this study suggest future research 41 should focus on practices associated with chewing betel, potential animal interactions including 42 pests in the home and pets, and risk factors associated with water. 44 AUTHOR SUMMARY 45 Since a new variant of chronic kidney disease was acknowledged in the early 1990s among those 46 in the agricultural community of Sri Lanka, especially rice farmers, the research community has bioRxiv preprint first posted online Nov. 10, 2019; doi: http://dx.doi.org/10.1101/837393. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. 47 searched for causes of what has become known as chronic kidney disease of unknown etiology or 48 CKDu. Previous studies have focused on heavy metals in the environment as they are known to be 49 toxic to the kidneys however, a proverbial “smoking gun” has yet to be identified. Understanding 50 that the causes is potential multifactorial we implemented a pilot case-control study using a One 51 Health methodology administering a comprehensive interview to assess environmental, animal, 52 and, human exposures that may be contributing to the diagnosis of CKDu. We found statistically 53 significant odds ratio among those that reported having a pet dog, chewing betel (a traditional 54 preparation or various ingredients wrapped in a betel leaf inserted between the teeth and cheek), 55 pests in the home, treating drinking water, and older age. These results serve to guide further 56 hypothesis generation regarding mechanisms behind associated exposures from infectious diseases 57 such as hantavirus and leptospirosis to food preparation through boiling drinking water in 58 aluminum vessels and oral pesticide exposure linked to betel preparation. 60 INTRODUCTION 61 There has been a notable increase in the recognized incidence of chronic kidney disease th 62 (CKD) around the world [1]. Kidney disease has moved from 27 most common cause of death in th 63 1990, to 18 in 2010 and has come to be considered a global public health problem causing high 64 morbidity, mortality, and financial burden [2-4]. Global prevalence of CKD is estimated to range 65 between 8% and 16%, and differs substantially across developed and developing countries [3, 5]. 66 Although diabetes mellitus and hypertension remain the leading causes of CKD, in recent years a 67 different form of CKD has reached epidemic levels, devastating rural communities in the dry zone 68 of Sri Lanka [6, 7]. The recognition of endemic CKD in the dry zone in the 1990s coincided with 69 the development of the rural healthcare system, which improved access to clinics by affected bioRxiv preprint first posted online Nov. 10, 2019; doi: http://dx.doi.org/10.1101/837393. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. 70 individuals. Since that time, the dry zone has seen a disproportionate increase in cases of CKD 71 compared to the rest of the country [8]. Existing studies describe the majority of these CKD 72 patients as not having hypertension or diabetes mellitus, two of the major risk factors for CKD. It 73 has therefore, been defined as a distinct condition: CKD of unknown or uncertain etiology 74 (CKDu). Similar chronic kidney disease hotspots have been recognized among farmers in Central 75 America (Nicaragua and El Salvador) and South Asia [9, 10]. 76 Approximately 2.5 million people live in the subset of Sri Lankan provinces where CKDu 77 is most common [11, 12]. Cases of this disease predominate in the Medawachchiya, Wilgamuwa, 78 Nikawewa, and Girandurukotte regions of the dry zone. Studies have shown the highest prevalence 79 of CKDu among 30-60 year old men engaged in chena or rice farming, and estimate a total of 80 20,000 (approximately 0.8% population) affected in the North Central Province [8, 13]. 81 The epidemic of CKDu in the dry zone is burdening the rural healthcare system and 82 impacting agricultural productivity due to a reduction in the available labor force when CKDu 83 patients are too ill to work [14-16]. Due to the irreversible and progressive nature of CKD, most 84 patients require long-term dialysis since renal transplants are not commonly available. For these 85 reasons, there is a need to determine the risk factors associated with CKDu to control and attenuate 86 the incidence of new CKDu cases. A growing body of evidence suggests that CKDu is multi- 87 factorial, making it difficult to identify individual risk factors and potential interactions involved 88 in pathogenesis [7, 12, 17-20]. Recently, various heavy metal agents such as cadmium, arsenic 89 agrochemicals, aluminum, and fluoride, as well as infectious diseases such as leptospirosis have 90 been considered for association with CKDu [21-26]. 91 Collaboration between researchers at the University of Peradeniya in Sri Lanka, the 92 University of California, Davis (UCD) in the United States, and Sri Lankan stakeholders in CKDu- bioRxiv preprint first posted online Nov. 10, 2019; doi: http://dx.doi.org/10.1101/837393. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. 93 endemic areas were involved in this pilot study. The driving hypothesis for this study is that alcohol 94 consumption and/or pesticide exposure are associated with CKDu as a health outcome. In addition, 95 it is recognized that relationships between key demographic, cultural, and occupational variables 96 may play a role in CKDu health outcomes [27-31]. 98 MATERIALS AND METHODS 99 A pilot case-control study was conducted in Sri Lanka from July-October 2015. The study 100 population was comprised of individuals (cases and controls) who resided in the North Central 101 Province (NCP) or Uva Province (UP) and sought medical care at Girandurukotte district hospital 102 (UP) or Medawachchiya clinic (NCP). The population in both the NCP and UP is approximately 103 1.2 million, with women making up the slight majority (51%) [13]. The majority of people in both 104 provinces are Sinhalese-speaking and resides in the rural sector where they engage in farming 105 (chena, rice). The NCP has the highest recorded prevalence of CKDu cases in Sri Lanka and is 106 located in the country’s dry zone. Uva Province is in the intermediate zone adjacent to the dry 107 zone, with a lower prevalence of CKDu cases compared to the NCP. 108 In order to test the hypothesis that there is a relationship between alcohol consumption and 109 CKDu diagnosis, and pesticide exposure and CKDu, in endemic areas of Sri Lanka, a questionnaire 110 survey was developed [See Additional File 1]. The survey tool encompassed a wide range of 111 exposures to capture potential unknown confounders, including exposures suggested by local 112 CKDu working groups at the University of Peradeniya. Individuals meeting the CKDu case 113 definitions as well as a comparison (control) population from the same endemic region were 114 invited for participation in the survey. 115 Case Definition bioRxiv preprint first posted online Nov. 10, 2019; doi: http://dx.doi.org/10.1101/837393. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. 116 Individuals diagnosed with definite or probable CKDu by a nephrologist at the 117 Girandurukotte regional hospital (GRH) or Medawachchiya renal clinic (MRC) made up the 118 population from which study cases were selected. An individual was considered a definite CKDu 119 case if creatinine levels were elevated and in subsequent renal biopsy the finding was predominant 120 tubular interstitial nephritis. A probable CKDu case was defined as persistent renal dysfunction 121 for more than 3 months after excluding known causes including hypertension, diabetes mellitus, 122 any other known renal diseases. This methodology is consistent with clinic/hospital programs and 123 represents the CKDu process of diagnosis in the region. Controls were chosen based on negative 124 results for CKDu from population screening records at GRH or MRC. 125 Recruitment 126 All controls were recruited using CKDu screening results within the past three years. These 127 CKDu negative were invited via post (hard copy letter) to return to the healthcare facility 128 associated with the previous screening to take part in a survey. Participation in the survey was 129 optional. All cases were recruited from Girandurukotte regional hospital or Medawachchiya renal 130 clinic. 131 Sample Size Calculation 132 The total sample size calculated for this pilot case-control study was 110, comprising 1:1 133 cases to controls. The target sample size of 110 individuals was calculated based on a power of 134 80% (β= 0.2), 95% confidence (α= 0.05), and a minimum effect size of 3.0. This relatively large 135 effect size was considered in the exploratory study in order to identify preliminary exposures 136 strongly associated with the outcome. An estimate of 26% was used for any reported alcohol 137 consumption among controls for the sample size calculation [32, 33]. 138 Survey Design bioRxiv preprint first posted online Nov. 10, 2019; doi: http://dx.doi.org/10.1101/837393. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. 139 Survey questions were designed by the research team in consultation with the resource 140 personnel at the Centre for Research, Education, and Training on Kidney Diseases (CERTkID) in 141 Sri Lanka prior to IRB approval, survey training, and interviews. The survey consisted of 138 142 questions structured as binary, categorical, ordinal, and open-ended across six categories: 1) 143 agricultural information; 2) animal exposure; 3) water and nutrition; 4) alcohol consumption; 5) 144 respondent demographics; and 6) family and past medical history. 145 The agricultural information section included questions related to farming practices and 146 agrochemical usage. Information on ownership and health of livestock and pets, presence of pest 147 animals and wild life were collected in section two of the survey. In the water and nutrition 148 information section, sources for drinking, cooking, and bathing water were assessed, along with 149 participant practices regarding water treatment prior to use. The alcohol consumption information 150 section contained questions on type of alcohol consumed, betel chewing and smoking status. 151 Alcohol consumption was assessed in two ways: a binary question was asked first on whether the 152 participant had ever consumed alcohol (if yes, what type and frequency) and second, whether the 153 participant believed that alcohol was a problem in their village. The respondent demographics 154 section contained questions pertaining to level of education and family income. To assess a 155 potential genetic component of CKDu, participants were asked whether their spouses were close 156 blood relatives and family history of CKDu, hypertension, and diabetes mellitus was also recorded. 157 Detailed survey and explanation of survey components are given in Supplementary information. 158 Survey data were collected by 12 trained graduate students associated with the University 159 of Peradeniya. In addition, investigators were present at each site during data collection, allowing 160 surveyors the opportunity for clarification as needed in real-time as interviews were conducted. 161 Cases and controls participated voluntarily, and surveys were administered verbally in the mother bioRxiv preprint first posted online Nov. 10, 2019; doi: http://dx.doi.org/10.1101/837393. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. 162 tongue of the participants (Sinhala) after obtaining consent (see Supplementary Information for 163 the English version of the consent form and survey questions). Survey question responses were 164 recorded on paper copies of the questionnaire by the surveyor. Each interview took approximately 165 one hour to complete. 166 The research protocol was designed according to the guidelines of the International 167 Compilation of Human Research Standards (2015 edition) and approved by the University of 168 California, Davis Institutional Review Board (#762486-2). Written consent of all study 169 participants was obtained by signature or thumbprint after survey enumerators verbally read the 170 consent statement in the appropriate language. The consent form was translated in both Sinhala 171 and Tamil. The majority of survey interviews took place at medical clinics specializing in renal 172 disease. 173 Statistical Analyses 174 Logistic regression was used to evaluate risk factors for CKDu case status. Bivariate 175 analysis was used to identify covariate with p-value ≤ 0.20 which was used to restrict consideration 176 for the final model. Pearson’s correlation coefficient was used to identify covariate correlation at - 177 0.5 ≤ 𝜌 ≥ 0.5. Data analysis was completed using Stata IC 14 (StataCorp. 2015. Stata Statistical 178 Software: Release 14. College Station, TX: StataCorp LP). Age was evaluated as a continuous 179 predictor and others were assessed as binary or categorical. Multiple logistic regression analyses 180 were performed using backwards stepwise selection to model the risk factors associated with the 181 CKDu disease outcome of interest. Adjusted analysis was done to control for possible confounding 182 by measured covariates. Statistical significance was assessed at the 𝛼 = 0.05. Any observations 183 missing data were restricted from the analysis dataset. All geospatial illustrations were prepared 184 using QGIS v2.18 (Free Software Foundation Inc., Boston MA, USA) with administrative bioRxiv preprint first posted online Nov. 10, 2019; doi: http://dx.doi.org/10.1101/837393. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. 185 boundaries provided by GADM v2.8 (Center for Spatial Statistic, University of California Davis, 186 USA, accessed November 2015). 188 RESULTS 189 A total of 110 participants were included in the analysis; 56 met the case definition and 54 190 satisfied control criteria (Table S1). All participants resided in the CKDu endemic regions in 191 Girandurukotte and Medawachchiya districts in Sri Lanka at the time of diagnosis. No missing 192 data was identified in the analysis dataset. Participants had a mean age of 52.6 years (range = 25- 193 80); there was a slight majority of males (60%) to females (40%). Most participants reported to be 194 married, with about half reporting being married to a spouse that was a close blood relative and 195 slightly over half reporting a family member having been diagnosed with CKDu (Table S1). Of 196 the 110 study participants, half reported consuming any type of alcohol and the majority reported 197 using some type of pesticide in their daily lives (insecticide, herbicide, in-home pesticide and/or 198 fungicide) (Table S1). 199 The majority of participants (74) were residing in the Uva Province. Twenty-two 200 participants resided in the North Central Province, five in the Eastern Province, two in the Central 201 Province and one resided in the Northwestern Province. There were 8 (7.3%) individuals for whom 202 reliable current residence information was not available due to survey legibility and 203 standardization complications. Data regarding province in which the participant was born were 204 collected (Fig 1). No participants reported birth outside of Sri Lanka. 206 Figure 1. Map of Sri Lanka by district indicating participant’s birth district bioRxiv preprint first posted online Nov. 10, 2019; doi: http://dx.doi.org/10.1101/837393. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. 207 After bivariate and correlational analyses, smoking unfiltered cigarettes and smoking 208 cannabis were found to be highly correlated (correlation coefficient = 0.5141 > 0.5 threshold). 209 Smoking unfiltered cigarettes was dropped from model consideration given a bivariate p-value 210 higher than that of smoking cannabis (p-value = 0.186 and 0.112, respectively). 211 Sources of drinking water were surveyed and ‘dug well’ was the most common source 212 (90.9%) of drinking/cooking water, with ‘rainwater collection containers’ being the second most 213 common source. Drinking water was reported to be treated with routine methods such as boiling 214 or filtering, or traditional methods such as placing igini (strychnos potatorum) seeds in the water 215 source/well [34]. A subset of key population characteristics is reported in Table 1. 216 Table 1: Study Population Characteristics by Case-Control Status Factor Level Control Case p-value N 54 56 Age, mean (SD)* 49.5 (11.7) 57.5 (9.6) <0.001 Female 27 (51) 16 (29) Gender* 0.017 Male 26 (49) 40 (71) Farming as No 14 (28) 3 (6) 0.002 Occupation* Yes 36 (72) 51 (94) Drinking Water Dug Well 48 (89) 52 (93) 0.47 Source Rain Water 6 (11) 5 (9) 0.7 Treat Drinking Water Drinking 34 (63) 41 (75) 0.19 Keep Livestock Livestock 17 (31) 19 (34) 0.78 Tobacco 8 (15) 14 (25) 0.18 Smoking Status Cannabis 2 (4) 7 (12) 0.092 Chew Betel* Betel 22 (41) 40 (71) 0.001 Not a Problem 13 (27) 11 (21) Alcohol a Problem in Minor Problem 17 (35) 20 (38) 0.85 Village Moderate Problem 10 (20) 9 (17) Major Problem 9 (18) 12 (23) Alcohol Consumption Any Alcohol 22 (41) 33 (59) 0.056 bioRxiv preprint first posted online Nov. 10, 2019; doi: http://dx.doi.org/10.1101/837393. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. Arrack 19 (35) 27 (48) 0.17 Beer 14 (26) 16 (29) 0.76 Kasippu 13 (24) 18 (32) 0.35 Any 42 (78) 52 (93) 0.025 Fungicide 21 (44) 29 (55) 0.27 Pesticide Use* Herbicide 41 (76) 51 (91) 0.032 Insecticide 42 (78) 44 (79) 0.92 217 *Significance attributed as p-value < 0.05 218 There is a significant difference between age, gender, occupation, chewing betel, pesticide 219 use at the 95% confidence level. The cases were older, on average, by eight years than the control 220 group with a gender imbalance finding males more often among the cases compared to controls. 221 Cases cited farming as an occupation more often than controls (22% higher among cases), reported 222 chewing betel (30% higher among cases), and used pesticides more often, specifically herbicide, 223 (15% higher among cases). 224 Two multivariable models were constructed, an exposure of interest model and an 225 exploratory model. The exposure of interest model forced inclusion of variables concerning 226 alcohol consumption and pesticide exposure, as neither exposure of interest was found to be 227 significantly associated with CKDu in bivariate analysis (Table 2). The final exposure of interest 228 model included four variables and excluded one variable compared to the stepwise method used 229 for the exploratory model (Table 3). The exploratory model (Table 3) included only risk factors 230 significantly associated with CKDu status (P<0.05). The primary exposures of interest (alcohol 231 consumption and pesticide exposure) were not found to be significant using a backward stepwise 232 selection process. However, age – considered as a continuous variable (OR: 1.08, 95% CI 1.02, 233 1.13), chewing betel (OR 4.01, 95% CI 1.49, 10.81), keeping a pet dog (OR: 4.21, 95% CI 1.55, 234 11.48), and reporting pests in the home (OR: 3.96, 95% CI 1.21, 12.93) were significantly 235 associated with CKDu case status. bioRxiv preprint first posted online Nov. 10, 2019; doi: http://dx.doi.org/10.1101/837393. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. 236 Table 2. Factors Associated with CKDu including Pesticide and Alcohol Consumption in 237 Sri Lanka Factor Odds Ratio P-Value 95% Confidence Interval Age* 1.12 <0.01 1.04 1.21 Sex: Male 6.19 0.07 0.73 44.06 Chew betel 3.57 0.08 0.86 14.84 Pet dog* 4.41 0.03 1.19 16.27 Pests in-home* 8.19 0.02 1.45 46.18 Consume Arrack 0.64 0.62 0 .11 3.86 Consume Beer 1.15 0.88 0.18 7.34 Consume Kasippu 0.26 0.23 0.03 2.39 Fungicide 1.45 0.57 0.41 5.17 Herbicide 1.00 1.00 - - In-home pesticide 0.82 0.79 0.34 4.97 Insecticide 1.00 1.00 - - 239 Note: Exposures of interest kept in model despite non-significant p-value 240 *Significance attributed as p-value < 0.05 242 Table 3. Factors Associated with CKDu in Sri Lanka Factor Odds Ratio P-Value 95% Confidence Interval Chew betel* 5.95 0.002 1.878 18.856 Pet dog* 3.515 0.012 1.312 9.42 Treat water* 3.944 0.026 1.175 13.236 Pests in-home* 5.708 0.009 1.538 21.182 Age*† 1.078 0.003 1.025 1.133 bioRxiv preprint first posted online Nov. 10, 2019; doi: http://dx.doi.org/10.1101/837393. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. *Significance attributed as p-value < 0.05; † Odds ratio per one year age increase 243 Bivariate analyses for specific types of pesticides and fertilizers used are detailed in Table 244 4. Before adjustment for age, gender, occupation, and alcohol consumption, usage of a fertilizer 245 (muriate of potash) and an herbicide (glyphosate) were significantly associated with confirmed 246 CKDu cases. However, after adjustment, none of the fertilizers, insecticides, or herbicides reported 247 were found to be significantly associated with the outcome of interest, and so were not included in 248 multivariable models (Table 4). 249 Table 4. Agrochemical Association with CKDu - Crude and Adjusted Odds Ratios Control Case Crude Odds Adjusted Odds Factor (N=54) (N=56) Ratio (95% CI) Ratio (95% CI) n(%) n(%) Fertilizer Urea 38 (70%) 47 (84%) 2.20 (0.9, 5.5) 0.92 (0.3, 3.2) Muriate of Potash* 1 (2%) 0 (0%) 3.15 (1.4, 7.4) 1.86 (0.7, 5.1) Triple Super Phosphate 11 (20%) 21 (38%) 2.35 (1.0, 5.5) 1.84 (0.7, 5.2) Mud/Manure 11 (20%) 5 (9%) 0.38 (0.1, 1.2) 0.41 (0.1, 1.5) Insecticide Carbosulfan 5 (9%) 3 (5%) 0.55 (0.13, 2.44) 0.48 (0.1, 2.5) Carbofuran 4 (7%) 4 (7%) 0.96 (0.23, 4.06) 0.47 (0.1, 2.4) Curateer 7 (13%) 6 (11%) 0.81 (0.25, 2.57) 0.74 (0.2, 2.8) Herbicide Glyphosate* 19 (35%) 32 (57%) 2.46 (1.14, 5.30) 1.09 (0.4, 2.8) MCPA 24 (44%) 28 (50%) 1.25 (0.59, 2.65) 0.92 (0.4, 2.3) DPA 11 (20%) 17 (30%) 1.70 (0.71, 4.08) 0.88 (0.3, 2.5) bioRxiv preprint first posted online Nov. 10, 2019; doi: http://dx.doi.org/10.1101/837393. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. Metamifop 8 (15%) 8 (14%) 0.96 (0.33, 2.77) 1.39 (0.4, 4.9) 250 * p ≤0.05; Adjusted by age, gender, farming occupation and alcohol consumption 251 Figure 2: Proportional Venn Diagrams Representing Reported Alcohol Type Consumed by 252 Case-Control Status 253 Figure 2 illustrates the difference between cases and control by type of alcohol reportedly 254 consumed. Cases were more likely to report drinking. The overlap between all three types of 255 alcohol indicates that if one consumes alcohol it is common to drink all three types surveyed. 256 Arrack was the most commonly reported alcohol consumed across cases and controls. Overall 257 reported alcohol consumption was observed among 2.3% of women, while 81.8% of men 258 reported drinking alcohol (two-sided Fisher’s exact: <0.001). 259 DISCUSSION 260 There was no significant association detected between CKDu and pesticide exposure nor 261 alcohol consumption. However, there were significant associations identified for chewing betel, 262 owning a pet dog, treatment of drinking water, reporting pests in the home, and age. These 263 significant exposures provide insight into previously unconsidered routes and mechanism for 264 CKDu in addition to potential guidance on how to reduce odds of CKDu diagnosis. Chewing betel 265 could be a risk directly or indirectly through contamination of the traditional chew ingredients or 266 through handling and/or preparation of the betel chew. The association between report of a pet dog 267 could suggest a zoonotic pathway and pests in the home could indicate pest extermination agent 268 risks or a disease reservoir. Treatment of drinking water, especially boiling, may contribute to 269 consumption of aluminum with nephrotoxic consequences. The wide variety of associated 270 exposures suggests that there may be multiple risk factors associated with CKDu, which is 271 consistent with results of previous studies [35, 36]. bioRxiv preprint first posted online Nov. 10, 2019; doi: http://dx.doi.org/10.1101/837393. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. 272 It is important to note that those cases reporting ever having been diagnosed with diabetes 273 or hypertension were diagnosed after meeting the CKDu case definition. Prevalence of diabetes 274 among controls was high (24.1%) relative to the 2011 and 2030 national estimates of 7.8% and 275 9.1%, respectively [37]. The questionnaire did not differentiate between type I or type II diabetes 276 mellitus, limiting inferences about reasons for this discrepancy in prevalence. It is possible that 277 changes in meal preparation (bivariate OR = 1.60 95% CI 0.74-3.44) and/or food stuffs available 278 in rural Sri Lanka also play a role in the increased prevalence of diabetes in this control sample. 279 Conversely, diagnosis of hypertension is significantly higher (Fisher’s exact = 0.002) among the 280 cases. However, this is common sequelae of chronic kidney disease [38-40]. 281 Uses of a variety of agrochemicals are common throughout the farming dry zone regions 282 and are often readily available through government subsidies. In our study, only herbicide use was 283 shown to be significant in the bivariate analysis among all insecticide, fungicide, and in-home 284 pesticide parameters. It is possible that exposure to pesticide occurs among those farmers reporting 285 no use of pesticides on their crops through adjacent farm pesticide use in tandem with dynamic 286 environmental factors, i.e. flooding, water source contamination, and winds. 287 The ingredients used in making kasippu, an illicit locally brewed alcohol, were of special 288 interest in this study, due to prior hypothesis that pesticides are introduced in the brewing process. 289 However, study participants who reported drinking kasippu most often purchased it from other 290 villagers and either did not know the ingredients used or did not want to report drinking kasippu 291 due to it being an illicit form of alcohol in Sri Lanka as well as the perceived cultural stigma for 292 reporting use. Of those that did report drinking kasippu, some reported urea as an ingredient in 293 kasippu production, which could have potentially toxic biologic effects, leading to increased blood 294 urea nitrogen subsequently impacting kidney function [41, 42]. bioRxiv preprint first posted online Nov. 10, 2019; doi: http://dx.doi.org/10.1101/837393. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. 295 Previous studies have suggested that drinking water quality and contamination may be 296 associated with CKDu [43-45]. Prior studies have identified cases and controls on the basis of 297 groundwater source and found much larger odds of disease in males who drank water from shallow 298 wells, compared to males who drank from natural springs (OR 5.48 95% CI 3.46-8.66) [46, 47]. 299 Similar findings were found for women drinking from shallow wells (OR 4.40 95% CI 2.23-8.68) 300 [46]. Due to the broad application of pesticides in all aspects of farming, potentially nephrotoxic 301 pesticide agents contaminating the drinking water cannot be ruled out. Our findings differ 302 somewhat from these prior studies in the minor difference between 89% (controls) and 93% (cases) 303 reported source of drinking water as a dug well. We however, were not able to compare other 304 sources of drinking water with high confidence and statistical power. 305 Dug wells are traditional wells often lined with clay brick and may be covered to prevent 306 animals from entering. There were few participants who received water via a tap line, rainwater 307 collectors, or methods other than a dug well. As such, our ability to evaluate drinking water source 308 as a risk factor was limited. In addition, the number of years that participants used different types 309 of drinking water was inconsistently recorded. Treatment of drinking water was found to be a 310 significant risk factor for CKDu. Treatment of water included boiling water (n=41), filtering water 311 (n=59), and traditional methods (n=19). The most common traditional practice for water treatment 312 was the introduction of Strychnos potatorum seeds (Sinhalese - ingini seeds) into the water source, 313 as is customary in Sri Lanka and India [48]. One possible risk for developing CKDu related to 314 treating drinking water could be the boiling (n = 41) of water in aluminum vessels [49]. 315 Information regarding the type of cookware used with relation to boiling water was not collected. 316 Our study found novel risk factors for CKDu in the study region. Results regarding the 317 potential mechanisms of association with CKDu for chewing betel, treatment of drinking water, bioRxiv preprint first posted online Nov. 10, 2019; doi: http://dx.doi.org/10.1101/837393. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. 318 and having pets or pests are inconclusive. For instance, dogs in Sri Lanka are often community 319 pets and as such, ownership can be difficult to ascertain, although 43% of study participants 320 reported having a pet dog. Additionally, dogs may serve as reservoirs of infectious disease that 321 may lead to renal damage in humans. For example, outbreaks of leptospirosis have been reported 322 during the monsoon season in some CKDu endemic regions [50, 51]. Leptospirosis causes acute 323 kidney failure in dogs, with high mortality rates among those that are untreated or without access 324 to dialysis. Consequently, dogs may potentially shed Leptospira spirochetes in their urine. Owners 325 or community individuals may be thus exposed, potentially clearing the infection but suffering 326 renal damage that could later lead to developing CKDu [52]. Current Ministry of Health CKDu 327 diagnosis criteria do not include serology or polymerase chain reaction (PCR) tests for ruling out 328 leptospirosis; patients are asked only to self-report previous history of leptospirosis. As 329 leptospirosis diagnostics continue to improve, future research should consider specimen collection 330 for laboratory confirmation [53-55]. 331 Mammalian pests in households may also be carriers of infectious disease that leads to 332 increased susceptibility to CKDu. There is an emerging hypothesis that Hanta virus could be the 333 possible causative agent for CKDu in Sri Lanka’s dry zone [26, 56]. Humans infected with Hanta 334 virus show clinical signs similar to those of Leptospirosis, and Hanta virus infection in humans 335 was first described in Sri Lanka in the mid-eighties [57]. Rodents act as the reservoir host for Hanta 336 virus and Leptospirosis. Therefore, our study may provide further evidence for the association 337 between infected rodents and prevalence of CKDu in Sri Lanka. Further research should evaluate 338 the incidence of Hanta virus and Leptospirosis in rodents, pets, livestock, and people in CKDu- 339 endemic regions in Sri Lanka. bioRxiv preprint first posted online Nov. 10, 2019; doi: http://dx.doi.org/10.1101/837393. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. 340 To identify other disease risks that might be due to close contact with domestic animals or 341 wildlife, data was collected concerning participant observations of illness among domestic animals 342 in the community. There were very few reported, including three observations of ill cattle (2.7%), 343 one goat (0.9%), and one chicken (0.9%) in the month prior to the survey interview. It is possible 344 that the lifespan of dogs and livestock in the community is short due to environmental hazards 345 (disease, trauma, etc.) and animals go missing or die before disease can be detected or transmitted 346 to humans. 347 Chewing betel was another novel risk factor for CKDu in our study. This practice is quite 348 common among Sri Lankans, and our study found that chewing betel was more common among 349 those reporting farming as an occupation (60%) than other occupations (40%). There is evidence 350 that betel preparations include stimulant properties similar to nicotine, and chewing it routinely 351 can lead to enamel erosion and oral cancer [58-60]. The betel preparation commonly chewed in 352 Sri Lanka is comprised of betel, areca nut, tobacco, and lime. However, betel recipes among 353 farmers in Sri Lanka’s dry zone may contain differing substances compared to preparations in the 354 remainder of the country, which could be associated with CKDu [58, 61, 62]. Individuals that mix 355 and distribute pesticides may be at greater risk, as betel is inserted in the mouth and may be done 356 in the field where hand washing is not possible. In addition, there is evidence that chewing betel 357 increases exposure to arsenic and cadmium, both of which can be nephrotoxic [63]. 358 Additionally, at our sample size (n=110), we do not have the power to detect small 359 differences in effect measure or to generalize to populations outside our study region. Finally, 360 although survey questions were detailed in nature, the question interpretation by the study 361 participant may have varied, leading to inaccurate answers. Due to the condensed nature of the 362 survey timeline, with multiple interviews occurring at one time, investigators could not oversee bioRxiv preprint first posted online Nov. 10, 2019; doi: http://dx.doi.org/10.1101/837393. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. 363 each individual interview. As such, details pertaining to the number of years when specific 364 pesticides or water sources were used were sometimes incomplete. 365 This case-control study design was useful for having comparable populations with and 366 without the disease in order to efficiently evaluate past risk factors associated with disease status. 367 In the future, a cohort study would be a useful design to evaluate exposure data, exposure timelines, 368 and incidence rates for CKDu, and we recommend that future studies of CKDu in Sri Lanka be 369 cohort-based, despite the longer follow-up period and greater expense. In addition, a nationwide, 370 coordinated CKDu research consortium spanning all major research institutions would make 371 CKDu research more efficient by standardizing study design and methodologies. This would allow 372 more accurate conclusions to be drawn from studies with clear and consistent case/control 373 definitions and study locations. 374 While our survey was comprehensive, our study had several limitations, primarily relating 375 to case definition, disease progression, and study design characteristics. Temporal bias might have 376 been introduced since the exact date of CKDu diagnosis was not collected however, the impact of 377 bias on alcohol consumption is believed to be minimal due to a non-significant difference in the 378 mean years since first drink (t = -1.89) and frequency of alcohol consumption (t = -0.49). 379 Furthermore, there was not a significant difference in the number of years farming among those 380 reporting a farming occupation (Fisher’s exact = 0.07) between cases and control. 381 The appearance and progression of CKDu can involve non-specific symptoms, making the 382 disease challenging to diagnose in the early, pre-clinical stages limiting the population of cases in 383 this study to those that were in advanced stages of the disease. Studies suggest using more sensitive 384 methods for detecting early CKDu, with measurement of microalbuminuria and functional markers 385 such as Cyst C, creatinine or tubular proteins like RBP4, NGAL or KIM would be beneficial in bioRxiv preprint first posted online Nov. 10, 2019; doi: http://dx.doi.org/10.1101/837393. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. 386 capturing a greater number of early-stage CKDu cases so that exposures can be better assessed and 387 treatment initiated earlier [64-66]. 388 The lack of association between herbicide and CKDu outcome in the adjusted model 389 indicates either that herbicide alone is not responsible for CKDu, or that sufficient detail regarding 390 herbicide use was not captured in the survey. For example, the volume and length of use of 391 pesticides was difficult to assess in an interview setting compared to a household visit, where 392 farmers could reference pesticide receipts or other family members for details they could not recall. 393 More detailed responses regarding usage may have resulted in a difference between cases and 394 controls, which could not be evaluated in this study. 395 At present, the use of the albumin-creatinine ratio or persistent proteinuria as an initial 396 screening tool is only sensitive at detecting advanced-stage prevalent CKDu cases. This could 397 cause misclassification of the disease outcome if the diagnostic test sensitivity is not high enough 398 to differentiate between early-stage CKDu patients and controls. 400 CONCLUSION 401 In conclusion, this pilot case-control study showed that chewing betel, reporting of in-home pets 402 and pests, treating drinking water, and age were significantly associated with CKDu. These 403 cultural and environmental factors are likely part of a multi-factorial etiology that is challenging 404 to unravel, and that may take years to understand whether preventive measures are effective. Future 405 studies should be cohort in design and focus on further exploring the identified risk factors and 406 their epidemiologic relationships to CKDu, as well as possible interventions to attenuate the 407 incidence of CKDu in Sri Lanka. Potential interventions to be considered based on these findings 408 might include safe home pest control options, testing and treatment for leptospirosis among bioRxiv preprint first posted online Nov. 10, 2019; doi: http://dx.doi.org/10.1101/837393. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. 409 community dogs, routine chronic kidney disease screening among those in CKDu endemic areas, 410 and education focused on hand hygiene in the field. These findings should be considered as 411 research regarding CKDu in other endemic regions continues. 413 DECLARATIONS 415 Ethics approval and consent to participate 416 The study was approved by the University of California, Davis, Institutional Review 417 Board (#762486-2). All participants voluntarily provided written consent prior to questionnaire 418 administration in their preferred language (Sinhala or Tamil). 420 Consent for publication 421 No personal, identifiable information is depicted nor represented. 423 Availability of Data and Material 424 Full questionnaire made available in additional file 1. 426 Competing interests 427 There are no competing nor conflicts of interest to report. 428 Funding 429 Funding for this research was made possible through a research PASS grant from the 430 Blum Center for Developing Economies at UC Davis, and a summer research fellowship award 431 from the University of California Global Health Institute. bioRxiv preprint first posted online Nov. 10, 2019; doi: http://dx.doi.org/10.1101/837393. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. 433 Author’s contributions 434 All authors listed in this manuscript meet the International Committee of Journal Editors 435 criteria for authorship. 437 Acknowledgements 438 Data collection for this study was made possible through the support provided by the 439 Centre for Research, Education, and Training on Kidney Diseases (CERTkID), namely by the late 440 Mr. Ranjith Mulleriya. Twelve graduate students of University of Peradeniya contributed to the 441 study by interviewing the cases and controls. David Bunn, Ph.D. and Michael Wilkes, M.D. 442 contributed to the researchers’ understanding of the cultural role of alcohol and pesticide use. 443 REFERENCES 444 1. 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The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. 649 learned from the WHO CKDu population prevalence study. BMC Nephrol 2014, 650 15:125. 651 66. Ratnayake S, Badurdeen Z, Nanayakkara N, Abeysekara T, Ratnatunga N, Kumarasiri 652 R: Screening for chronic kidney disease of uncertain aetiology in Sri Lanka: 653 usability of surrogate biomarkers over dipstick proteinuria. BMC Nephrol 2017, 654 18(1):199. 657 APPENDIX: 658 Table S1: Comprehensive Participant Characteristics by Exposure Category 660 SUPPORTING INFORMATION LEGENDS 661 Figure 1: Map of Sri Lanka by district identifying jurisdiction in which survey 662 participant was born. Map produced using QGIS v2.18 (Free Software Foundation Inc. Boston, 663 MA). Jurisdiction boundaries as specified by GADM v2.8, Center for Spatial Statistic, 664 University of California, Davis, California, USA, accessed November 2015. 665 https://gadm.org/data.html. 666 Figure 2: Proportional Venn Diagram produced using Stata IC 14 (Stata Corp., College 667 Station, TX). 668 Table S1: Characteristics of Population Surveyed: exhaustive list of characteristics 669 among survey participants by exposure category. bioRxiv preprint first posted online Nov. 10, 2019; doi: http://dx.doi.org/10.1101/837393. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. bioRxiv preprint first posted online Nov. 10, 2019; doi: http://dx.doi.org/10.1101/837393. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png bioRxiv bioRxiv

A pilot study of behavioral, environmental, and occupational risk factors for chronic kidney disease of unknown etiology in Sri Lanka

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Abstract

bioRxiv preprint first posted online Nov. 10, 2019; doi: http://dx.doi.org/10.1101/837393. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. 1 Title: A pilot study of behavioral, environmental, and occupational risk factors for chronic 2 kidney disease of unknown etiology in Sri Lanka 4 Short Title: Chronic kidney disease of unknown etiology (CKDu) case-control study in Sri 5 Lanka 1,2 3 3,4 5 6 7 Jake M. Pry ; Wendi Jackson ; Ruwini Rupasinghe ; Guneratne Lishanthe ; Zied Badurdeen , 6 7 3 4 8 Tilak Abeysekara ; Rohana Chandrajith ; Woutrina Smith , Saumya Wickramasinghe 10 Centre for Infectious Disease Research Zambia, Lusaka, Zambia 11 School of Medicine, Washington University, St. Louis, MO, U.S.A. 12 School of Veterinary Medicine, University of California, Davis, U.S.A 13 Faculty of Veterinary Medicine and Animal Science, University of Peradeniya, Sri Lanka 14 Renal Research Centre, District Hospital, Girandurukotte, Sri Lanka 15 Faculty of Medicine, University of Peradeniya, Sri Lanka 16 Faculty of Science, University of Peradeniya, Sri Lanka 18 Corresponding author: jpry@wustl.edu 19 Jake Michael Pry 20 Department of Infectious Disease 21 Washington University 22 660 S. Euclid Ave. St. Louis, MO 63110 23 Mobile. +260 96 3088750 | +1 936-661-6885 bioRxiv preprint first posted online Nov. 10, 2019; doi: http://dx.doi.org/10.1101/837393. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. 24 ABSTRACT 25 Chronic kidney disease of unknown etiology (CKDu) was first recognized in Sri Lanka in the early 26 1990s, and since then it has reached epidemic levels in the North Central Province of the country. 27 The prevalence of CKDu is reportedly highest among communities that engage in chena and paddy 28 farming, which is most often practiced in the dry zone including the North Central and East Central 29 Provinces of Sri Lanka. Previous studies have suggested varied hypotheses for the etiology of 30 CKDu; however, there is not yet a consensus on the primary risk factors, possibly due to disparate 31 study designs, sample populations, and methodologies. The goal of this pilot case-control study 32 was to evaluate the relationships between key demographic, cultural, and occupational variables 33 as risk factors for CKDu, with a primary interest in pesticide exposure both occupationally and 34 through its potential use as an ingredient in brewed kasippu alcohol. A total of 56 CKDu cases 35 and 54 control individuals were surveyed using a proctored, self-reported questionnaire. 36 Occupational pesticide exposure and alcohol consumption were not found to be significant risk 37 factors for CKDu. However, a statistically significant association with CKDu was observed with 38 chewing betel (OR: 6.11, 95% CI: 1.93, 19.35), age (OR: 1.07, 95% CI: 1.02, 1.13), owning a pet 39 dog (OR: 3.74, 95% CI: 1.38, 10.11), water treatment (OR: 3.68, 95% CI: 1.09, 12.43) and pests 40 in the house (OR: 5.81, 95% CI: 1.56, 21.60). The findings of this study suggest future research 41 should focus on practices associated with chewing betel, potential animal interactions including 42 pests in the home and pets, and risk factors associated with water. 44 AUTHOR SUMMARY 45 Since a new variant of chronic kidney disease was acknowledged in the early 1990s among those 46 in the agricultural community of Sri Lanka, especially rice farmers, the research community has bioRxiv preprint first posted online Nov. 10, 2019; doi: http://dx.doi.org/10.1101/837393. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. 47 searched for causes of what has become known as chronic kidney disease of unknown etiology or 48 CKDu. Previous studies have focused on heavy metals in the environment as they are known to be 49 toxic to the kidneys however, a proverbial “smoking gun” has yet to be identified. Understanding 50 that the causes is potential multifactorial we implemented a pilot case-control study using a One 51 Health methodology administering a comprehensive interview to assess environmental, animal, 52 and, human exposures that may be contributing to the diagnosis of CKDu. We found statistically 53 significant odds ratio among those that reported having a pet dog, chewing betel (a traditional 54 preparation or various ingredients wrapped in a betel leaf inserted between the teeth and cheek), 55 pests in the home, treating drinking water, and older age. These results serve to guide further 56 hypothesis generation regarding mechanisms behind associated exposures from infectious diseases 57 such as hantavirus and leptospirosis to food preparation through boiling drinking water in 58 aluminum vessels and oral pesticide exposure linked to betel preparation. 60 INTRODUCTION 61 There has been a notable increase in the recognized incidence of chronic kidney disease th 62 (CKD) around the world [1]. Kidney disease has moved from 27 most common cause of death in th 63 1990, to 18 in 2010 and has come to be considered a global public health problem causing high 64 morbidity, mortality, and financial burden [2-4]. Global prevalence of CKD is estimated to range 65 between 8% and 16%, and differs substantially across developed and developing countries [3, 5]. 66 Although diabetes mellitus and hypertension remain the leading causes of CKD, in recent years a 67 different form of CKD has reached epidemic levels, devastating rural communities in the dry zone 68 of Sri Lanka [6, 7]. The recognition of endemic CKD in the dry zone in the 1990s coincided with 69 the development of the rural healthcare system, which improved access to clinics by affected bioRxiv preprint first posted online Nov. 10, 2019; doi: http://dx.doi.org/10.1101/837393. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. 70 individuals. Since that time, the dry zone has seen a disproportionate increase in cases of CKD 71 compared to the rest of the country [8]. Existing studies describe the majority of these CKD 72 patients as not having hypertension or diabetes mellitus, two of the major risk factors for CKD. It 73 has therefore, been defined as a distinct condition: CKD of unknown or uncertain etiology 74 (CKDu). Similar chronic kidney disease hotspots have been recognized among farmers in Central 75 America (Nicaragua and El Salvador) and South Asia [9, 10]. 76 Approximately 2.5 million people live in the subset of Sri Lankan provinces where CKDu 77 is most common [11, 12]. Cases of this disease predominate in the Medawachchiya, Wilgamuwa, 78 Nikawewa, and Girandurukotte regions of the dry zone. Studies have shown the highest prevalence 79 of CKDu among 30-60 year old men engaged in chena or rice farming, and estimate a total of 80 20,000 (approximately 0.8% population) affected in the North Central Province [8, 13]. 81 The epidemic of CKDu in the dry zone is burdening the rural healthcare system and 82 impacting agricultural productivity due to a reduction in the available labor force when CKDu 83 patients are too ill to work [14-16]. Due to the irreversible and progressive nature of CKD, most 84 patients require long-term dialysis since renal transplants are not commonly available. For these 85 reasons, there is a need to determine the risk factors associated with CKDu to control and attenuate 86 the incidence of new CKDu cases. A growing body of evidence suggests that CKDu is multi- 87 factorial, making it difficult to identify individual risk factors and potential interactions involved 88 in pathogenesis [7, 12, 17-20]. Recently, various heavy metal agents such as cadmium, arsenic 89 agrochemicals, aluminum, and fluoride, as well as infectious diseases such as leptospirosis have 90 been considered for association with CKDu [21-26]. 91 Collaboration between researchers at the University of Peradeniya in Sri Lanka, the 92 University of California, Davis (UCD) in the United States, and Sri Lankan stakeholders in CKDu- bioRxiv preprint first posted online Nov. 10, 2019; doi: http://dx.doi.org/10.1101/837393. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. 93 endemic areas were involved in this pilot study. The driving hypothesis for this study is that alcohol 94 consumption and/or pesticide exposure are associated with CKDu as a health outcome. In addition, 95 it is recognized that relationships between key demographic, cultural, and occupational variables 96 may play a role in CKDu health outcomes [27-31]. 98 MATERIALS AND METHODS 99 A pilot case-control study was conducted in Sri Lanka from July-October 2015. The study 100 population was comprised of individuals (cases and controls) who resided in the North Central 101 Province (NCP) or Uva Province (UP) and sought medical care at Girandurukotte district hospital 102 (UP) or Medawachchiya clinic (NCP). The population in both the NCP and UP is approximately 103 1.2 million, with women making up the slight majority (51%) [13]. The majority of people in both 104 provinces are Sinhalese-speaking and resides in the rural sector where they engage in farming 105 (chena, rice). The NCP has the highest recorded prevalence of CKDu cases in Sri Lanka and is 106 located in the country’s dry zone. Uva Province is in the intermediate zone adjacent to the dry 107 zone, with a lower prevalence of CKDu cases compared to the NCP. 108 In order to test the hypothesis that there is a relationship between alcohol consumption and 109 CKDu diagnosis, and pesticide exposure and CKDu, in endemic areas of Sri Lanka, a questionnaire 110 survey was developed [See Additional File 1]. The survey tool encompassed a wide range of 111 exposures to capture potential unknown confounders, including exposures suggested by local 112 CKDu working groups at the University of Peradeniya. Individuals meeting the CKDu case 113 definitions as well as a comparison (control) population from the same endemic region were 114 invited for participation in the survey. 115 Case Definition bioRxiv preprint first posted online Nov. 10, 2019; doi: http://dx.doi.org/10.1101/837393. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. 116 Individuals diagnosed with definite or probable CKDu by a nephrologist at the 117 Girandurukotte regional hospital (GRH) or Medawachchiya renal clinic (MRC) made up the 118 population from which study cases were selected. An individual was considered a definite CKDu 119 case if creatinine levels were elevated and in subsequent renal biopsy the finding was predominant 120 tubular interstitial nephritis. A probable CKDu case was defined as persistent renal dysfunction 121 for more than 3 months after excluding known causes including hypertension, diabetes mellitus, 122 any other known renal diseases. This methodology is consistent with clinic/hospital programs and 123 represents the CKDu process of diagnosis in the region. Controls were chosen based on negative 124 results for CKDu from population screening records at GRH or MRC. 125 Recruitment 126 All controls were recruited using CKDu screening results within the past three years. These 127 CKDu negative were invited via post (hard copy letter) to return to the healthcare facility 128 associated with the previous screening to take part in a survey. Participation in the survey was 129 optional. All cases were recruited from Girandurukotte regional hospital or Medawachchiya renal 130 clinic. 131 Sample Size Calculation 132 The total sample size calculated for this pilot case-control study was 110, comprising 1:1 133 cases to controls. The target sample size of 110 individuals was calculated based on a power of 134 80% (β= 0.2), 95% confidence (α= 0.05), and a minimum effect size of 3.0. This relatively large 135 effect size was considered in the exploratory study in order to identify preliminary exposures 136 strongly associated with the outcome. An estimate of 26% was used for any reported alcohol 137 consumption among controls for the sample size calculation [32, 33]. 138 Survey Design bioRxiv preprint first posted online Nov. 10, 2019; doi: http://dx.doi.org/10.1101/837393. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. 139 Survey questions were designed by the research team in consultation with the resource 140 personnel at the Centre for Research, Education, and Training on Kidney Diseases (CERTkID) in 141 Sri Lanka prior to IRB approval, survey training, and interviews. The survey consisted of 138 142 questions structured as binary, categorical, ordinal, and open-ended across six categories: 1) 143 agricultural information; 2) animal exposure; 3) water and nutrition; 4) alcohol consumption; 5) 144 respondent demographics; and 6) family and past medical history. 145 The agricultural information section included questions related to farming practices and 146 agrochemical usage. Information on ownership and health of livestock and pets, presence of pest 147 animals and wild life were collected in section two of the survey. In the water and nutrition 148 information section, sources for drinking, cooking, and bathing water were assessed, along with 149 participant practices regarding water treatment prior to use. The alcohol consumption information 150 section contained questions on type of alcohol consumed, betel chewing and smoking status. 151 Alcohol consumption was assessed in two ways: a binary question was asked first on whether the 152 participant had ever consumed alcohol (if yes, what type and frequency) and second, whether the 153 participant believed that alcohol was a problem in their village. The respondent demographics 154 section contained questions pertaining to level of education and family income. To assess a 155 potential genetic component of CKDu, participants were asked whether their spouses were close 156 blood relatives and family history of CKDu, hypertension, and diabetes mellitus was also recorded. 157 Detailed survey and explanation of survey components are given in Supplementary information. 158 Survey data were collected by 12 trained graduate students associated with the University 159 of Peradeniya. In addition, investigators were present at each site during data collection, allowing 160 surveyors the opportunity for clarification as needed in real-time as interviews were conducted. 161 Cases and controls participated voluntarily, and surveys were administered verbally in the mother bioRxiv preprint first posted online Nov. 10, 2019; doi: http://dx.doi.org/10.1101/837393. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. 162 tongue of the participants (Sinhala) after obtaining consent (see Supplementary Information for 163 the English version of the consent form and survey questions). Survey question responses were 164 recorded on paper copies of the questionnaire by the surveyor. Each interview took approximately 165 one hour to complete. 166 The research protocol was designed according to the guidelines of the International 167 Compilation of Human Research Standards (2015 edition) and approved by the University of 168 California, Davis Institutional Review Board (#762486-2). Written consent of all study 169 participants was obtained by signature or thumbprint after survey enumerators verbally read the 170 consent statement in the appropriate language. The consent form was translated in both Sinhala 171 and Tamil. The majority of survey interviews took place at medical clinics specializing in renal 172 disease. 173 Statistical Analyses 174 Logistic regression was used to evaluate risk factors for CKDu case status. Bivariate 175 analysis was used to identify covariate with p-value ≤ 0.20 which was used to restrict consideration 176 for the final model. Pearson’s correlation coefficient was used to identify covariate correlation at - 177 0.5 ≤ 𝜌 ≥ 0.5. Data analysis was completed using Stata IC 14 (StataCorp. 2015. Stata Statistical 178 Software: Release 14. College Station, TX: StataCorp LP). Age was evaluated as a continuous 179 predictor and others were assessed as binary or categorical. Multiple logistic regression analyses 180 were performed using backwards stepwise selection to model the risk factors associated with the 181 CKDu disease outcome of interest. Adjusted analysis was done to control for possible confounding 182 by measured covariates. Statistical significance was assessed at the 𝛼 = 0.05. Any observations 183 missing data were restricted from the analysis dataset. All geospatial illustrations were prepared 184 using QGIS v2.18 (Free Software Foundation Inc., Boston MA, USA) with administrative bioRxiv preprint first posted online Nov. 10, 2019; doi: http://dx.doi.org/10.1101/837393. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. 185 boundaries provided by GADM v2.8 (Center for Spatial Statistic, University of California Davis, 186 USA, accessed November 2015). 188 RESULTS 189 A total of 110 participants were included in the analysis; 56 met the case definition and 54 190 satisfied control criteria (Table S1). All participants resided in the CKDu endemic regions in 191 Girandurukotte and Medawachchiya districts in Sri Lanka at the time of diagnosis. No missing 192 data was identified in the analysis dataset. Participants had a mean age of 52.6 years (range = 25- 193 80); there was a slight majority of males (60%) to females (40%). Most participants reported to be 194 married, with about half reporting being married to a spouse that was a close blood relative and 195 slightly over half reporting a family member having been diagnosed with CKDu (Table S1). Of 196 the 110 study participants, half reported consuming any type of alcohol and the majority reported 197 using some type of pesticide in their daily lives (insecticide, herbicide, in-home pesticide and/or 198 fungicide) (Table S1). 199 The majority of participants (74) were residing in the Uva Province. Twenty-two 200 participants resided in the North Central Province, five in the Eastern Province, two in the Central 201 Province and one resided in the Northwestern Province. There were 8 (7.3%) individuals for whom 202 reliable current residence information was not available due to survey legibility and 203 standardization complications. Data regarding province in which the participant was born were 204 collected (Fig 1). No participants reported birth outside of Sri Lanka. 206 Figure 1. Map of Sri Lanka by district indicating participant’s birth district bioRxiv preprint first posted online Nov. 10, 2019; doi: http://dx.doi.org/10.1101/837393. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. 207 After bivariate and correlational analyses, smoking unfiltered cigarettes and smoking 208 cannabis were found to be highly correlated (correlation coefficient = 0.5141 > 0.5 threshold). 209 Smoking unfiltered cigarettes was dropped from model consideration given a bivariate p-value 210 higher than that of smoking cannabis (p-value = 0.186 and 0.112, respectively). 211 Sources of drinking water were surveyed and ‘dug well’ was the most common source 212 (90.9%) of drinking/cooking water, with ‘rainwater collection containers’ being the second most 213 common source. Drinking water was reported to be treated with routine methods such as boiling 214 or filtering, or traditional methods such as placing igini (strychnos potatorum) seeds in the water 215 source/well [34]. A subset of key population characteristics is reported in Table 1. 216 Table 1: Study Population Characteristics by Case-Control Status Factor Level Control Case p-value N 54 56 Age, mean (SD)* 49.5 (11.7) 57.5 (9.6) <0.001 Female 27 (51) 16 (29) Gender* 0.017 Male 26 (49) 40 (71) Farming as No 14 (28) 3 (6) 0.002 Occupation* Yes 36 (72) 51 (94) Drinking Water Dug Well 48 (89) 52 (93) 0.47 Source Rain Water 6 (11) 5 (9) 0.7 Treat Drinking Water Drinking 34 (63) 41 (75) 0.19 Keep Livestock Livestock 17 (31) 19 (34) 0.78 Tobacco 8 (15) 14 (25) 0.18 Smoking Status Cannabis 2 (4) 7 (12) 0.092 Chew Betel* Betel 22 (41) 40 (71) 0.001 Not a Problem 13 (27) 11 (21) Alcohol a Problem in Minor Problem 17 (35) 20 (38) 0.85 Village Moderate Problem 10 (20) 9 (17) Major Problem 9 (18) 12 (23) Alcohol Consumption Any Alcohol 22 (41) 33 (59) 0.056 bioRxiv preprint first posted online Nov. 10, 2019; doi: http://dx.doi.org/10.1101/837393. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. Arrack 19 (35) 27 (48) 0.17 Beer 14 (26) 16 (29) 0.76 Kasippu 13 (24) 18 (32) 0.35 Any 42 (78) 52 (93) 0.025 Fungicide 21 (44) 29 (55) 0.27 Pesticide Use* Herbicide 41 (76) 51 (91) 0.032 Insecticide 42 (78) 44 (79) 0.92 217 *Significance attributed as p-value < 0.05 218 There is a significant difference between age, gender, occupation, chewing betel, pesticide 219 use at the 95% confidence level. The cases were older, on average, by eight years than the control 220 group with a gender imbalance finding males more often among the cases compared to controls. 221 Cases cited farming as an occupation more often than controls (22% higher among cases), reported 222 chewing betel (30% higher among cases), and used pesticides more often, specifically herbicide, 223 (15% higher among cases). 224 Two multivariable models were constructed, an exposure of interest model and an 225 exploratory model. The exposure of interest model forced inclusion of variables concerning 226 alcohol consumption and pesticide exposure, as neither exposure of interest was found to be 227 significantly associated with CKDu in bivariate analysis (Table 2). The final exposure of interest 228 model included four variables and excluded one variable compared to the stepwise method used 229 for the exploratory model (Table 3). The exploratory model (Table 3) included only risk factors 230 significantly associated with CKDu status (P<0.05). The primary exposures of interest (alcohol 231 consumption and pesticide exposure) were not found to be significant using a backward stepwise 232 selection process. However, age – considered as a continuous variable (OR: 1.08, 95% CI 1.02, 233 1.13), chewing betel (OR 4.01, 95% CI 1.49, 10.81), keeping a pet dog (OR: 4.21, 95% CI 1.55, 234 11.48), and reporting pests in the home (OR: 3.96, 95% CI 1.21, 12.93) were significantly 235 associated with CKDu case status. bioRxiv preprint first posted online Nov. 10, 2019; doi: http://dx.doi.org/10.1101/837393. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. 236 Table 2. Factors Associated with CKDu including Pesticide and Alcohol Consumption in 237 Sri Lanka Factor Odds Ratio P-Value 95% Confidence Interval Age* 1.12 <0.01 1.04 1.21 Sex: Male 6.19 0.07 0.73 44.06 Chew betel 3.57 0.08 0.86 14.84 Pet dog* 4.41 0.03 1.19 16.27 Pests in-home* 8.19 0.02 1.45 46.18 Consume Arrack 0.64 0.62 0 .11 3.86 Consume Beer 1.15 0.88 0.18 7.34 Consume Kasippu 0.26 0.23 0.03 2.39 Fungicide 1.45 0.57 0.41 5.17 Herbicide 1.00 1.00 - - In-home pesticide 0.82 0.79 0.34 4.97 Insecticide 1.00 1.00 - - 239 Note: Exposures of interest kept in model despite non-significant p-value 240 *Significance attributed as p-value < 0.05 242 Table 3. Factors Associated with CKDu in Sri Lanka Factor Odds Ratio P-Value 95% Confidence Interval Chew betel* 5.95 0.002 1.878 18.856 Pet dog* 3.515 0.012 1.312 9.42 Treat water* 3.944 0.026 1.175 13.236 Pests in-home* 5.708 0.009 1.538 21.182 Age*† 1.078 0.003 1.025 1.133 bioRxiv preprint first posted online Nov. 10, 2019; doi: http://dx.doi.org/10.1101/837393. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. *Significance attributed as p-value < 0.05; † Odds ratio per one year age increase 243 Bivariate analyses for specific types of pesticides and fertilizers used are detailed in Table 244 4. Before adjustment for age, gender, occupation, and alcohol consumption, usage of a fertilizer 245 (muriate of potash) and an herbicide (glyphosate) were significantly associated with confirmed 246 CKDu cases. However, after adjustment, none of the fertilizers, insecticides, or herbicides reported 247 were found to be significantly associated with the outcome of interest, and so were not included in 248 multivariable models (Table 4). 249 Table 4. Agrochemical Association with CKDu - Crude and Adjusted Odds Ratios Control Case Crude Odds Adjusted Odds Factor (N=54) (N=56) Ratio (95% CI) Ratio (95% CI) n(%) n(%) Fertilizer Urea 38 (70%) 47 (84%) 2.20 (0.9, 5.5) 0.92 (0.3, 3.2) Muriate of Potash* 1 (2%) 0 (0%) 3.15 (1.4, 7.4) 1.86 (0.7, 5.1) Triple Super Phosphate 11 (20%) 21 (38%) 2.35 (1.0, 5.5) 1.84 (0.7, 5.2) Mud/Manure 11 (20%) 5 (9%) 0.38 (0.1, 1.2) 0.41 (0.1, 1.5) Insecticide Carbosulfan 5 (9%) 3 (5%) 0.55 (0.13, 2.44) 0.48 (0.1, 2.5) Carbofuran 4 (7%) 4 (7%) 0.96 (0.23, 4.06) 0.47 (0.1, 2.4) Curateer 7 (13%) 6 (11%) 0.81 (0.25, 2.57) 0.74 (0.2, 2.8) Herbicide Glyphosate* 19 (35%) 32 (57%) 2.46 (1.14, 5.30) 1.09 (0.4, 2.8) MCPA 24 (44%) 28 (50%) 1.25 (0.59, 2.65) 0.92 (0.4, 2.3) DPA 11 (20%) 17 (30%) 1.70 (0.71, 4.08) 0.88 (0.3, 2.5) bioRxiv preprint first posted online Nov. 10, 2019; doi: http://dx.doi.org/10.1101/837393. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. Metamifop 8 (15%) 8 (14%) 0.96 (0.33, 2.77) 1.39 (0.4, 4.9) 250 * p ≤0.05; Adjusted by age, gender, farming occupation and alcohol consumption 251 Figure 2: Proportional Venn Diagrams Representing Reported Alcohol Type Consumed by 252 Case-Control Status 253 Figure 2 illustrates the difference between cases and control by type of alcohol reportedly 254 consumed. Cases were more likely to report drinking. The overlap between all three types of 255 alcohol indicates that if one consumes alcohol it is common to drink all three types surveyed. 256 Arrack was the most commonly reported alcohol consumed across cases and controls. Overall 257 reported alcohol consumption was observed among 2.3% of women, while 81.8% of men 258 reported drinking alcohol (two-sided Fisher’s exact: <0.001). 259 DISCUSSION 260 There was no significant association detected between CKDu and pesticide exposure nor 261 alcohol consumption. However, there were significant associations identified for chewing betel, 262 owning a pet dog, treatment of drinking water, reporting pests in the home, and age. These 263 significant exposures provide insight into previously unconsidered routes and mechanism for 264 CKDu in addition to potential guidance on how to reduce odds of CKDu diagnosis. Chewing betel 265 could be a risk directly or indirectly through contamination of the traditional chew ingredients or 266 through handling and/or preparation of the betel chew. The association between report of a pet dog 267 could suggest a zoonotic pathway and pests in the home could indicate pest extermination agent 268 risks or a disease reservoir. Treatment of drinking water, especially boiling, may contribute to 269 consumption of aluminum with nephrotoxic consequences. The wide variety of associated 270 exposures suggests that there may be multiple risk factors associated with CKDu, which is 271 consistent with results of previous studies [35, 36]. bioRxiv preprint first posted online Nov. 10, 2019; doi: http://dx.doi.org/10.1101/837393. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. 272 It is important to note that those cases reporting ever having been diagnosed with diabetes 273 or hypertension were diagnosed after meeting the CKDu case definition. Prevalence of diabetes 274 among controls was high (24.1%) relative to the 2011 and 2030 national estimates of 7.8% and 275 9.1%, respectively [37]. The questionnaire did not differentiate between type I or type II diabetes 276 mellitus, limiting inferences about reasons for this discrepancy in prevalence. It is possible that 277 changes in meal preparation (bivariate OR = 1.60 95% CI 0.74-3.44) and/or food stuffs available 278 in rural Sri Lanka also play a role in the increased prevalence of diabetes in this control sample. 279 Conversely, diagnosis of hypertension is significantly higher (Fisher’s exact = 0.002) among the 280 cases. However, this is common sequelae of chronic kidney disease [38-40]. 281 Uses of a variety of agrochemicals are common throughout the farming dry zone regions 282 and are often readily available through government subsidies. In our study, only herbicide use was 283 shown to be significant in the bivariate analysis among all insecticide, fungicide, and in-home 284 pesticide parameters. It is possible that exposure to pesticide occurs among those farmers reporting 285 no use of pesticides on their crops through adjacent farm pesticide use in tandem with dynamic 286 environmental factors, i.e. flooding, water source contamination, and winds. 287 The ingredients used in making kasippu, an illicit locally brewed alcohol, were of special 288 interest in this study, due to prior hypothesis that pesticides are introduced in the brewing process. 289 However, study participants who reported drinking kasippu most often purchased it from other 290 villagers and either did not know the ingredients used or did not want to report drinking kasippu 291 due to it being an illicit form of alcohol in Sri Lanka as well as the perceived cultural stigma for 292 reporting use. Of those that did report drinking kasippu, some reported urea as an ingredient in 293 kasippu production, which could have potentially toxic biologic effects, leading to increased blood 294 urea nitrogen subsequently impacting kidney function [41, 42]. bioRxiv preprint first posted online Nov. 10, 2019; doi: http://dx.doi.org/10.1101/837393. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. 295 Previous studies have suggested that drinking water quality and contamination may be 296 associated with CKDu [43-45]. Prior studies have identified cases and controls on the basis of 297 groundwater source and found much larger odds of disease in males who drank water from shallow 298 wells, compared to males who drank from natural springs (OR 5.48 95% CI 3.46-8.66) [46, 47]. 299 Similar findings were found for women drinking from shallow wells (OR 4.40 95% CI 2.23-8.68) 300 [46]. Due to the broad application of pesticides in all aspects of farming, potentially nephrotoxic 301 pesticide agents contaminating the drinking water cannot be ruled out. Our findings differ 302 somewhat from these prior studies in the minor difference between 89% (controls) and 93% (cases) 303 reported source of drinking water as a dug well. We however, were not able to compare other 304 sources of drinking water with high confidence and statistical power. 305 Dug wells are traditional wells often lined with clay brick and may be covered to prevent 306 animals from entering. There were few participants who received water via a tap line, rainwater 307 collectors, or methods other than a dug well. As such, our ability to evaluate drinking water source 308 as a risk factor was limited. In addition, the number of years that participants used different types 309 of drinking water was inconsistently recorded. Treatment of drinking water was found to be a 310 significant risk factor for CKDu. Treatment of water included boiling water (n=41), filtering water 311 (n=59), and traditional methods (n=19). The most common traditional practice for water treatment 312 was the introduction of Strychnos potatorum seeds (Sinhalese - ingini seeds) into the water source, 313 as is customary in Sri Lanka and India [48]. One possible risk for developing CKDu related to 314 treating drinking water could be the boiling (n = 41) of water in aluminum vessels [49]. 315 Information regarding the type of cookware used with relation to boiling water was not collected. 316 Our study found novel risk factors for CKDu in the study region. Results regarding the 317 potential mechanisms of association with CKDu for chewing betel, treatment of drinking water, bioRxiv preprint first posted online Nov. 10, 2019; doi: http://dx.doi.org/10.1101/837393. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. 318 and having pets or pests are inconclusive. For instance, dogs in Sri Lanka are often community 319 pets and as such, ownership can be difficult to ascertain, although 43% of study participants 320 reported having a pet dog. Additionally, dogs may serve as reservoirs of infectious disease that 321 may lead to renal damage in humans. For example, outbreaks of leptospirosis have been reported 322 during the monsoon season in some CKDu endemic regions [50, 51]. Leptospirosis causes acute 323 kidney failure in dogs, with high mortality rates among those that are untreated or without access 324 to dialysis. Consequently, dogs may potentially shed Leptospira spirochetes in their urine. Owners 325 or community individuals may be thus exposed, potentially clearing the infection but suffering 326 renal damage that could later lead to developing CKDu [52]. Current Ministry of Health CKDu 327 diagnosis criteria do not include serology or polymerase chain reaction (PCR) tests for ruling out 328 leptospirosis; patients are asked only to self-report previous history of leptospirosis. As 329 leptospirosis diagnostics continue to improve, future research should consider specimen collection 330 for laboratory confirmation [53-55]. 331 Mammalian pests in households may also be carriers of infectious disease that leads to 332 increased susceptibility to CKDu. There is an emerging hypothesis that Hanta virus could be the 333 possible causative agent for CKDu in Sri Lanka’s dry zone [26, 56]. Humans infected with Hanta 334 virus show clinical signs similar to those of Leptospirosis, and Hanta virus infection in humans 335 was first described in Sri Lanka in the mid-eighties [57]. Rodents act as the reservoir host for Hanta 336 virus and Leptospirosis. Therefore, our study may provide further evidence for the association 337 between infected rodents and prevalence of CKDu in Sri Lanka. Further research should evaluate 338 the incidence of Hanta virus and Leptospirosis in rodents, pets, livestock, and people in CKDu- 339 endemic regions in Sri Lanka. bioRxiv preprint first posted online Nov. 10, 2019; doi: http://dx.doi.org/10.1101/837393. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. 340 To identify other disease risks that might be due to close contact with domestic animals or 341 wildlife, data was collected concerning participant observations of illness among domestic animals 342 in the community. There were very few reported, including three observations of ill cattle (2.7%), 343 one goat (0.9%), and one chicken (0.9%) in the month prior to the survey interview. It is possible 344 that the lifespan of dogs and livestock in the community is short due to environmental hazards 345 (disease, trauma, etc.) and animals go missing or die before disease can be detected or transmitted 346 to humans. 347 Chewing betel was another novel risk factor for CKDu in our study. This practice is quite 348 common among Sri Lankans, and our study found that chewing betel was more common among 349 those reporting farming as an occupation (60%) than other occupations (40%). There is evidence 350 that betel preparations include stimulant properties similar to nicotine, and chewing it routinely 351 can lead to enamel erosion and oral cancer [58-60]. The betel preparation commonly chewed in 352 Sri Lanka is comprised of betel, areca nut, tobacco, and lime. However, betel recipes among 353 farmers in Sri Lanka’s dry zone may contain differing substances compared to preparations in the 354 remainder of the country, which could be associated with CKDu [58, 61, 62]. Individuals that mix 355 and distribute pesticides may be at greater risk, as betel is inserted in the mouth and may be done 356 in the field where hand washing is not possible. In addition, there is evidence that chewing betel 357 increases exposure to arsenic and cadmium, both of which can be nephrotoxic [63]. 358 Additionally, at our sample size (n=110), we do not have the power to detect small 359 differences in effect measure or to generalize to populations outside our study region. Finally, 360 although survey questions were detailed in nature, the question interpretation by the study 361 participant may have varied, leading to inaccurate answers. Due to the condensed nature of the 362 survey timeline, with multiple interviews occurring at one time, investigators could not oversee bioRxiv preprint first posted online Nov. 10, 2019; doi: http://dx.doi.org/10.1101/837393. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. 363 each individual interview. As such, details pertaining to the number of years when specific 364 pesticides or water sources were used were sometimes incomplete. 365 This case-control study design was useful for having comparable populations with and 366 without the disease in order to efficiently evaluate past risk factors associated with disease status. 367 In the future, a cohort study would be a useful design to evaluate exposure data, exposure timelines, 368 and incidence rates for CKDu, and we recommend that future studies of CKDu in Sri Lanka be 369 cohort-based, despite the longer follow-up period and greater expense. In addition, a nationwide, 370 coordinated CKDu research consortium spanning all major research institutions would make 371 CKDu research more efficient by standardizing study design and methodologies. This would allow 372 more accurate conclusions to be drawn from studies with clear and consistent case/control 373 definitions and study locations. 374 While our survey was comprehensive, our study had several limitations, primarily relating 375 to case definition, disease progression, and study design characteristics. Temporal bias might have 376 been introduced since the exact date of CKDu diagnosis was not collected however, the impact of 377 bias on alcohol consumption is believed to be minimal due to a non-significant difference in the 378 mean years since first drink (t = -1.89) and frequency of alcohol consumption (t = -0.49). 379 Furthermore, there was not a significant difference in the number of years farming among those 380 reporting a farming occupation (Fisher’s exact = 0.07) between cases and control. 381 The appearance and progression of CKDu can involve non-specific symptoms, making the 382 disease challenging to diagnose in the early, pre-clinical stages limiting the population of cases in 383 this study to those that were in advanced stages of the disease. Studies suggest using more sensitive 384 methods for detecting early CKDu, with measurement of microalbuminuria and functional markers 385 such as Cyst C, creatinine or tubular proteins like RBP4, NGAL or KIM would be beneficial in bioRxiv preprint first posted online Nov. 10, 2019; doi: http://dx.doi.org/10.1101/837393. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. 386 capturing a greater number of early-stage CKDu cases so that exposures can be better assessed and 387 treatment initiated earlier [64-66]. 388 The lack of association between herbicide and CKDu outcome in the adjusted model 389 indicates either that herbicide alone is not responsible for CKDu, or that sufficient detail regarding 390 herbicide use was not captured in the survey. For example, the volume and length of use of 391 pesticides was difficult to assess in an interview setting compared to a household visit, where 392 farmers could reference pesticide receipts or other family members for details they could not recall. 393 More detailed responses regarding usage may have resulted in a difference between cases and 394 controls, which could not be evaluated in this study. 395 At present, the use of the albumin-creatinine ratio or persistent proteinuria as an initial 396 screening tool is only sensitive at detecting advanced-stage prevalent CKDu cases. This could 397 cause misclassification of the disease outcome if the diagnostic test sensitivity is not high enough 398 to differentiate between early-stage CKDu patients and controls. 400 CONCLUSION 401 In conclusion, this pilot case-control study showed that chewing betel, reporting of in-home pets 402 and pests, treating drinking water, and age were significantly associated with CKDu. These 403 cultural and environmental factors are likely part of a multi-factorial etiology that is challenging 404 to unravel, and that may take years to understand whether preventive measures are effective. Future 405 studies should be cohort in design and focus on further exploring the identified risk factors and 406 their epidemiologic relationships to CKDu, as well as possible interventions to attenuate the 407 incidence of CKDu in Sri Lanka. Potential interventions to be considered based on these findings 408 might include safe home pest control options, testing and treatment for leptospirosis among bioRxiv preprint first posted online Nov. 10, 2019; doi: http://dx.doi.org/10.1101/837393. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. 409 community dogs, routine chronic kidney disease screening among those in CKDu endemic areas, 410 and education focused on hand hygiene in the field. These findings should be considered as 411 research regarding CKDu in other endemic regions continues. 413 DECLARATIONS 415 Ethics approval and consent to participate 416 The study was approved by the University of California, Davis, Institutional Review 417 Board (#762486-2). All participants voluntarily provided written consent prior to questionnaire 418 administration in their preferred language (Sinhala or Tamil). 420 Consent for publication 421 No personal, identifiable information is depicted nor represented. 423 Availability of Data and Material 424 Full questionnaire made available in additional file 1. 426 Competing interests 427 There are no competing nor conflicts of interest to report. 428 Funding 429 Funding for this research was made possible through a research PASS grant from the 430 Blum Center for Developing Economies at UC Davis, and a summer research fellowship award 431 from the University of California Global Health Institute. bioRxiv preprint first posted online Nov. 10, 2019; doi: http://dx.doi.org/10.1101/837393. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. 433 Author’s contributions 434 All authors listed in this manuscript meet the International Committee of Journal Editors 435 criteria for authorship. 437 Acknowledgements 438 Data collection for this study was made possible through the support provided by the 439 Centre for Research, Education, and Training on Kidney Diseases (CERTkID), namely by the late 440 Mr. Ranjith Mulleriya. Twelve graduate students of University of Peradeniya contributed to the 441 study by interviewing the cases and controls. David Bunn, Ph.D. and Michael Wilkes, M.D. 442 contributed to the researchers’ understanding of the cultural role of alcohol and pesticide use. 443 REFERENCES 444 1. 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The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. 649 learned from the WHO CKDu population prevalence study. BMC Nephrol 2014, 650 15:125. 651 66. Ratnayake S, Badurdeen Z, Nanayakkara N, Abeysekara T, Ratnatunga N, Kumarasiri 652 R: Screening for chronic kidney disease of uncertain aetiology in Sri Lanka: 653 usability of surrogate biomarkers over dipstick proteinuria. BMC Nephrol 2017, 654 18(1):199. 657 APPENDIX: 658 Table S1: Comprehensive Participant Characteristics by Exposure Category 660 SUPPORTING INFORMATION LEGENDS 661 Figure 1: Map of Sri Lanka by district identifying jurisdiction in which survey 662 participant was born. Map produced using QGIS v2.18 (Free Software Foundation Inc. Boston, 663 MA). Jurisdiction boundaries as specified by GADM v2.8, Center for Spatial Statistic, 664 University of California, Davis, California, USA, accessed November 2015. 665 https://gadm.org/data.html. 666 Figure 2: Proportional Venn Diagram produced using Stata IC 14 (Stata Corp., College 667 Station, TX). 668 Table S1: Characteristics of Population Surveyed: exhaustive list of characteristics 669 among survey participants by exposure category. bioRxiv preprint first posted online Nov. 10, 2019; doi: http://dx.doi.org/10.1101/837393. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. bioRxiv preprint first posted online Nov. 10, 2019; doi: http://dx.doi.org/10.1101/837393. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.

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