Study design and population
A community based cross-sectional study was conducted in patients attending to a traditional Medicine clinic for CKDu in the Ayurveda hospital at Kebithigollewa for a period of six months in 2019. Geographically, this study site is flat area and the mean annual rainfall and temperature ranges between 1200 - 2000 mm and 20 oC to 32 oC respectively. The majority of the inhabitants in this area were farmers and the education level of the majority of adults was very low [15].
The population for the study involved patients who have been regularly visiting to the clinic and previously diagnosed as unknown form of CKD known as CKDu. These patients were treated with Sri Lankan traditional medicines. Ingredients and their actions use for Sri Lankan traditional medicine treatments are given in the supplementary file.
Patients with serum creatinine concentration above 1.2 mg/dl for a duration of more than six months were recruited for the study. The patients had pre-existing conditions of diabetes mellitus and hypertension were excluded from the study. In addition, patients with past history of pyelonephritis, glomerulonephritis, renal calculi or snake bite, on treatment for diabetes, increased glycosylated haemoglobin (HbA1c; >6.5%) and on treatment for hypertension; if not on treatment for hypertension, blood pressure above >160/100 mmHg were excluded from the study. Age, gender and race matched 20 healthy individuals from the same community with less than 1.2 mg/dl and had no previous reports of renal diseases were participated as controls.
Data Collection
The convenience sampling approach of non-probability was used to choose study participants. A validated interviewer-administered questionnaire was used to collect data. All participants were made aware of the study's purpose, and those who agreed to volunteer for it received an informational sheet and an informed consent form. The questionnaire was initially created in English before being back translated into Sri Lankans' native languages, Sinhala and Tamil.
Data collection was carried out by the field research team comprising the Western and Ayurvedic doctors, nurses, medical laboratory technologists and research assistants with the assistance of Public Health Midwife’s (field health staff). Research teams were trained prior to the study on all aspects of data collection. All subjects were made aware that participating was completely voluntary, that there was no risk involved with the treatment, and that their identities would be held in strictly confidential. Informed written consent forms were obtained from all participants and all information was kept in confidence.
Age, gender, occupation, and family history of CKDu were among the socio-demographic information on the patient that was documented. Participants independently choose whether to participate in the study, with no influence from the researchers. The participants completed the questionnaires without any interruption to their duties.
At every stage of the investigation, confidentiality was ensured. In order to ensure confidentiality, participant names were not listed on the questionnaires, and instead, each participant was assigned an index number. All of the gathered data were kept on a password-protected electronic device, and after five years, it will be permanently deleted.
Physical Measurements
Height of each participant was measured to the closest 0.1 cm, as the maximum distance from their heels to the top of their heads while they were standing barefoot and fully inspired (SECA model 240). Participants wore indoor light clothing and had their body weight measured with an SECA digital weighing scale to the closest 0.1 kg. BMI was calculated as weight in kilograms divided by height squared in meters (kgm-2). Two readings of blood pressure were taken at an interval of 10 minutes. Blood pressure was measured using a digital sphygmomanometer (Omron Auto BP - HEM 7322) 10 minutes after rest before blood sampling. The figure-of-eight technique was be used to measure ankle edema [16].
Sample collection
A total of 5 ml Blood was collected from each participant by qualified nurses following the standard universal precautions. Blood was centrifuged and serum separated in the field and stored in ice until transported to the laboratory for biochemical investigations.
A 5-10 ml of urine was collected from the patients into clean urine containers and sealed and stored at a temperature below 4ºC. Patients were given instructions on how to take low-contamination urine samples midstream. Samples were analyzed using MALB-KIT and QR-100 specific protein analyzers by a qualified laboratory technician. A specimen of blood (5ml) and urine were collected from each participant in one month interval for a period of six months.
Biochemical analysis
Serum creatinine was analyzed using modified Jaffe reaction that is traceable to a reference method based on isotope-dilution mass spectrometry (IDMS). eGFR values were estimated according to CKD-EPI equation using the available serum creatinine results [17].
An early morning mid-stream urine sample from everyone was collected for dipstick urinalysis for blood and protein, which would be read using an optical reader, and for measurement of urine albumin: creatinine ratio (ACR). These tests were conducted monthly for CKDu patients. Within 24 hours of collecting the urine sample, the levels of urine albumin were determined using an automated immunoturbidimetric technique. All blood and urine samples were analyzed in the accredited laboratory under the supervision of a biochemist.
Data Analysis
Data was entered into a Microsoft Excel sheet and transferred to 20th version of Statistical Package for the Social Sciences (SPSS). The socio-demographic variables were categorized and analysed utilizing variable analysis in the use of descriptive statistics. Then the data were differentiated based on the changes in standard ordinal measures such as count, percentage, mean and standard error of mean (SEM). Values for demographic variables, anthropometric measurements and bio chemical parameters of the study participants were reported as means with standard error of mean (SEM). Normality tests were applied and nonparametric tests were used in this study since values of biochemical and anthropometrical parameters were not normally distributed.
CKDu phases were staged using e-GFR based on CKD-EPI. Disease progression was determined according to the advance of CKDu stages over the monitoring period and changing pattern of monthly averages of variables. Scatter plots were used to evaluate the relationship between serum creatinine & microalbumin and e-GFR & microalbumin over a consecutive period of six months. Biochemical parameters were analyzed using spearman correlation test to determine the correlations between biochemical parameters. Multistate Markov models were used to estimate the rates of transition between stages of chronic diseases. In the present study, 3 stages (stage 3, stage 4 and stage 5) were considered for Multistate Markov models. Once a patient is in a particular stage, he can either move to another stage or stay back in the same stage. This stage movements of the patient will be decided only by his current stage and not the sequence of past stages because the current state has the power to predict the next stage. The level of significance for all statistical tests was considered as p-value was less than 0.05.