There was no significant association detected between CKDu and pesticide exposure nor alcohol consumption. However, there were significant associations identified for chewing betel, owning a pet dog, treatment of drinking water, reporting pests in the home, and age. These significant exposures provide insight into previously unconsidered routes and mechanism for CKDu in addition to potential guidance on how to reduce odds of CKDu diagnosis. Chewing betel could be a risk directly or indirectly through contamination of the traditional chew ingredients or through handling and/or preparation of the betel chew. The association between report of a pet dog could suggest a zoonotic pathway and pests in the home could indicate pest extermination agent risks or a disease reservoir. Treatment of drinking water, especially boiling, may contribute to consumption of aluminum with nephrotoxic consequences. The wide variety of associated exposures suggests that there may be multiple risk factors associated with CKDu, which is consistent with results of previous studies [37, 38].
It is important to note that those cases reporting ever having been diagnosed with diabetes or hypertension were diagnosed after meeting the CKDu case definition. Prevalence of diabetes among controls was high (24.1%) relative to the 2011 and 2030 national estimates of 7.8% and 9.1%, respectively . The questionnaire did not differentiate between type I or type II diabetes mellitus, limiting inferences about reasons for this discrepancy in prevalence. It is possible that changes in meal preparation (OR = 1.60 95% CI: 0.74-3.44) and/or food stuffs available in rural Sri Lanka also play a role in the increased prevalence of diabetes in this control sample. Conversely, diagnosis of hypertension is significantly higher (Fisher’s exact = 0.002) among the cases. However, this is common sequelae of chronic kidney disease [40-42].
Uses of a variety of agrochemicals are common throughout the farming dry zone regions and are often readily available through government subsidies. In our study, only herbicide use was shown to be significant in the bivariate analysis among all insecticide, fungicide, and in-home pesticide parameters. It is possible that exposure to pesticide occurs among those farmers reporting no use of pesticides on their crops through adjacent farm pesticide use in tandem with dynamic environmental factors, i.e. flooding, water source contamination, and winds.
The ingredients used in making kasippu, an illicit locally brewed alcohol, were of special interest in this study, due to prior hypothesis that pesticides are introduced in the brewing process. However, study participants who reported drinking kasippu most often purchased it from other villagers and either did not know the ingredients used or did not want to report drinking kasippu due to it being an illicit form of alcohol in Sri Lanka as well as the perceived cultural stigma for reporting use. Of those that did report drinking kasippu, some reported urea as an ingredient in kasippu production, which could have potentially toxic biologic effects, leading to increased blood urea nitrogen subsequently impacting kidney function [43, 44].
Previous studies have suggested that drinking water quality and contamination may be associated with CKDu [45-47]. Prior studies have identified cases and controls on the basis of groundwater source and found much larger odds of disease in males who drank water from shallow wells, compared to males who drank from natural springs (OR 5.48 95% CI: 3.46-8.66) [48, 49]. Similar findings were found for women drinking from shallow wells (OR 4.40 95% CI: 2.23-8.68) . Due to the broad application of pesticides in all aspects of farming, potentially nephrotoxic pesticide agents contaminating the drinking water cannot be ruled out. Our findings differ somewhat from these prior studies in the minor difference between 89% (controls) and 93% (cases) reported source of drinking water as a dug well. We however, were not able to compare other sources of drinking water with high confidence and statistical power.
Dug wells are traditional wells often lined with clay brick and may be covered to prevent animals from entering. There were few participants who received water via a tap line, rainwater collectors, or methods other than a dug well. As such, our ability to evaluate drinking water source as a risk factor was limited. In addition, the number of years that participants used different types of drinking water was inconsistently recorded. Treatment of drinking water was found to be a significant risk factor for CKDu. Treatment of water included boiling water (n=41), filtering water (n=59), and traditional methods (n=19). The most common traditional practice for water treatment was the introduction of Strychnos potatorum seeds (Sinhalese - ingini seeds) into the water source, as is customary in Sri Lanka and India . One possible risk for developing CKDu related to treating drinking water could be the boiling (n = 41) of water in aluminum vessels . Information regarding the type of cookware used with relation to boiling water was not collected.
Results regarding the potential mechanisms of association with CKDu for chewing betel, treatment of drinking water, and having pets or pests are inconclusive. However, it is possible that ownership of dogs in Sri Lanka, often community pets, may be difficult to ascertain, although 43% of study participants reported having a pet dog. These community dogs may serve as reservoirs of infectious disease such as leptospirosis. Outbreaks of leptospirosis have been reported during the monsoon season in some CKDu endemic regions [52, 53]. Consequently, dogs may potentially shed Leptospira spirochetes in their urine leading to community exposure. Humans infected with Leptospira through canine urine might passively clear the infection and experience renal damage that could later lead/contribute to the developments of CKDu . Current Ministry of Health CKDu diagnosis criteria do not include serology or polymerase chain reaction (PCR) tests for ruling out leptospirosis; patients are asked only to self-report previous history of leptospirosis. As leptospirosis diagnostics continue to improve, future research should consider specimen collection for laboratory confirmation [55-57].
Mammalian pests in households may also be carriers of infectious disease that leads to increased susceptibility to CKDu. There is an emerging hypothesis that Hanta virus could be the possible causative agent for CKDu in Sri Lanka’s dry zone [30, 58]. Humans infected with Hanta virus show clinical signs similar to those of Leptospirosis, and Hanta virus infection in humans was first described in Sri Lanka in the mid-eighties . Rodents act as the reservoir host for Hanta virus and Leptospira. Our study, therefore, may provide further evidence for the mechanism of increased CKDu prevalence through rodent pests and pet(s) serving as a potential route/intermediate for Hanta virus and/or Leptospira in humans in Sri Lanka. Further research should evaluate the prevalence of Hanta virus and Leptospira in rodents, pets, livestock, and people in CKDu-endemic regions in Sri Lanka. Additionally, Leptospira vaccination campaigns may be considered a potential intervention model to address CKDu.
To identify other disease risks that might be due to close contact with domestic animals or wildlife, data was collected concerning participant observations of illness among domestic animals in the community. There were very few reported, including three observations of ill cattle (2.7%), one goat (0.9%), and one chicken (0.9%) in the month prior to the survey interview. It is possible that the lifespan of dogs and livestock in the community is short due to environmental hazards (disease, trauma, etc.) and animals go missing or die before disease can be detected or transmitted to humans.
Chewing betel was another novel risk factor for CKDu in our study. This practice is quite common among Sri Lankans, and our study found that chewing betel was more common among those reporting farming as an occupation (60%) than other occupations (40%). There is evidence that betel preparations include stimulant properties similar to nicotine, and chewing it routinely can lead to enamel erosion and oral cancer [60-62]. The betel preparation commonly chewed in Sri Lanka is comprised of betel, areca nut, tobacco, and lime. However, betel recipes among farmers in Sri Lanka’s dry zone may contain differing substances compared to preparations in the remainder of the country, which could be associated with CKDu [60, 63, 64]. Individuals that mix and distribute pesticides may be at greater risk, as betel is inserted in the mouth and may be done in the field where hand washing is not possible. In addition, there is evidence that chewing betel increases exposure to arsenic and cadmium, both of which can be nephrotoxic .
Additionally, at our sample size (n=110), we do not have the power to detect small differences in effect measure or to generalize to populations outside our study region. Finally, although survey questions were detailed in nature, the question interpretation by the study participant may have varied, leading to inaccurate answers. Due to the condensed nature of the survey timeline, with multiple interviews occurring at one time, investigators could not oversee each individual interview. As such, details pertaining to the number of years when specific pesticides or water sources were used were sometimes incomplete.
This case-control study design was useful for having comparable populations with and without the disease in order to efficiently evaluate past risk factors associated with disease status. In the future, a cohort study would be a useful design to evaluate exposure data, exposure timelines, and incidence rates for CKDu, and we recommend that future studies of CKDu in Sri Lanka be cohort-based, despite the longer follow-up period and greater expense. In addition, a nationwide, coordinated CKDu research consortium spanning all major research institutions would make CKDu research more efficient by standardizing study design and methodologies. This would allow more accurate conclusions to be drawn from studies with clear and consistent case/control definitions and study locations.
While our survey was comprehensive, our study had several limitations, primarily relating to case definition, disease progression, and study design characteristics. Temporal bias might have been introduced since the exact date of CKDu diagnosis was not collected however, the impact of bias on alcohol consumption is believed to be minimal due to a non-significant difference in the mean years since first drink (t = -1.89) and frequency of alcohol consumption (t = -0.49). Furthermore, there was not a significant difference in the number of years farming among those reporting a farming occupation (Fisher’s exact = 0.07) between cases and control.
The appearance and progression of CKDu can involve non-specific symptoms, making the disease challenging to diagnose in the early, pre-clinical stages limiting the population of cases in this study to those that were in advanced stages of the disease. This may have caused the inclusion of false-negatives, sub-clinical cases, in our control group however, without further testing the extent of this issue is unknown. It is possible that survivor bias also played a role in the control group selection making the control group more resistant to the outcome however, the given the age of the control group this effect is likely small. Additionally, there is potentially some historical exposures that increase CKDu diagnosis potential given the younger mean age of control participants. Studies suggest using more sensitive methods for detecting early CKDu, with measurement of microalbuminuria and functional markers such as cystatin C, creatinine or tubular proteins like RBP4, NGAL or KIM would be beneficial in capturing a greater number of early-stage CKDu cases so that exposures can be better assessed and treatment initiated earlier [66-68].
The lack of association between herbicide and CKDu outcome in the adjusted model indicates either that herbicide alone is not responsible for CKDu, or that sufficient detail regarding herbicide use was not captured in the survey. For example, the volume and length of use of pesticides was difficult to assess in an interview setting compared to a household visit, where farmers could reference pesticide receipts or other family members for details they could not recall. More detailed responses regarding usage may have resulted in a difference between cases and controls, which could not be evaluated in this study.
At present, the use of the albumin-creatinine ratio or persistent proteinuria as an initial screening tool has limited capacity to detect CKDu cases before advanced-stage(s). This could cause misclassification of the disease outcome if the diagnostic test sensitivity is not high enough to differentiate between early-stage CKDu patients and controls.