CKDu Research Symposium Abstract Book

Sri Lanka Medical Association

World Kidney Day 2012

Research Symposium on Chronic Kidney Disease of Unknown Aetiology (CKDu)

Sunday, 11 March 2012, 8.30 am to 2.00 pm

Lionel Memorial Auditorium, Wijerama House, 6 Wijerama Mawatha, Colombo 7

SLMA­Research Symposium on CKDu

Message from the President, SLMA It gives me great pleasure to welcome all the resource persons, researchers, and delegates for this very important research symposium on chronic kidney disease of unknown aetiology (CKDu) prevalent in several parts of our country.

This symposium is a result of a request made by the Director General of Health Services, from the SLMA to provide a common platform for researchers working in this field to present their research findings so that one could come to evidenced based conclusions about the aetiology of CKDu. I am pleased that the SLMA was able to organise this symposium in a very short period of time, so that it could be held on 8 March 2012, the World Kidney Day. I thank all the resource persons who agreed readily to make presentations, the researchers who sent in abstracts for their enthusiasm to present their research, and the delegates for their enthusiastic participation.

I hope that at the end of the day, we could come to some meaningful evidence based conclusions about CKDu that we could present to the relevant authorities.

Prof. Vajira H. W. Dissanayake President Sri Lanka Medical Association

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SLMA­Research Symposium on CKDu

PROGRAMME 08.30 am

Registration

08.45 am

Welcome Address Prof. Vajira H. W. Dissanayake, President, SLMA

08.50 am

Introductory Remarks Prof. Rezvi Sheriff Senior Professor of Clinical Medicine Faculty of Medicine, University of Colombo

09.00 am

09.30 am

The role played by the Ministry of Health in tackling CKDu Dr. Palitha Maheepala Additional Secretary Ministry of Health Chairperson, Research Coordination Committee for CKDu Presentation of Research Findings EPIDEMIOLOGY OF CKDu Dissanayake DM1, Jayasekera JMKB1, Ranasinghe AV2 1Department of Pathology, Faculty of Medicine, University of Peradeniya; 2Health Department, North Central Province, Anuradhapura. Sri Lanka. dhammika62@gmail.com

A STUDY OF GEOGRAPHICAL DISTRIBUTION OF CHRONIC KIDNEY DISEASE OF UNKNOWN ORIGIN IN SRI LANKA Dissanayake DM1 , Jayasekara JMKB1 , Adhikari SB2, Bandara P3 1Department of Pathology, Faculty of Medicine, University of Peradeniya; 2Mahaweli Development Authority, Kotmale, 3Health Department, North Central Province, Anuradhapura. dhammika62@gmail.com

CHRONIC KIDNEY DISEASE OF UNCERTAIN AETIOLOGY PREVALENT IN SRI LANKA Athuruliya NTC, Abeysekara TDJ, Amerasinghe PH, Kumarasiri R, Bandara P, Karunarathne U Department of Clinical Pharmacology, Faculty of Medicine, University of Peradeniya; Nephrology Unit, Teaching Hospital Kandy; International Water Management Institute, Colombo; Department of Community Medicine, University of Peradeniya; Ministry of Health, Colombo. ckdeupdnsl@yahoo.com

EPIDEMIOLOGICAL RISK FACTORS OF CHRONIC KIDNEY DISEASE OF UNKNOWN AETIOLOGY: A HOSPITAL BASED CASE CONTROL STUDY Chandrakumara WAJC1, Jankan N5, Marystella M3, Perera K4, Pushpakumara PHGJ2, Ruparathne AA1, Rupasinghe S2, Thilakaratne YGRKK1 1Department of Paediatrics, 2 Department of Psychiatry, Faculty of Medicine & Allied Sciences, Rajarata University; 3Postgraduate Institute of Science, 4Department of Engineering Mathematics, Faculty of Engineering, University of Peradeniya; 5Epidemiolgy Unit. janakatechno@yahoo.com 2

SLMA­Research Symposium on CKDu

OCCUPATIONAL RISK FACTORS & RISK INDEX FOR CHRONIC KIDNEY DISEASE OF UNKNOWN AETIOLOGY IN SRI LANKA Jayasekara JMKB1, Dissanayake1 DM, Gunaratna MDN2, Thilakarathna S3 1 Department of Pathology, Faculty of Medicine, University of Peradeniya; 2Department of Mathematics, Faculty of Engineering, University of Moratuwa; 3 Department of Medical Laboratory Sciences, Faculty of Allied Health Sciences, University of Peradeniya. dhammika62@gmail.com

RISK FACTORS ASSOCIATED WITH CHRONIC KIDNEY DISEASE OF UNKNOWN AETIOLOGY

Wanigasuriya KP1, Peiris‐John RJ2,3, Wickremasinghe R4 1Department of Medicine, University of Sri Jayewardenepura, Nugegoda; 2Department of Physiology, University of Sri Jayewardenepura, Nugegoda; 3Section of Epidemiology and Biostatistics, University of Auckland, Auckland, New Zealand; 4Department of Public health, University of Kelaniya, Ragama, kamaniw@sltnet.lk

11.00 am

11.15 am

URINARY β2­MICROGLOBULIN VARIATION BETWEEN CHRONIC KIDNEY DISEASE PATIENTS OF UNKNOWN AETIOLOGY AND NON AFFECTED CONTROLS Siriwardhana EARIE 1, Perera PAJ1, Sivakanesan R2, Abeysekara T3 1Department of Biochemistry, Faculty of Medicine and Allied Sciences, Rajarata University; 2Department of Biochemistry, Faculty of Medicine, University of Peradeniya; 3Department of Pharmacology, Faculty of Medicine, University of Peradeniya. rangaedirisinghe@yahoo.co.uk

RENAL TUBULAR FUNCTIONS OF FARMERS OF HIGH PREVALENCE AREA FOR CKDu Dissananayake DM, Jayasekera JMKB, Wimalasiri R, Ratnayake I, Dissanayake S Department of Pathology, Faculty of Medicine, University of Peradeniya. adhammika62@gmail.com

CORRELATION BETWEEN THE ULTRA SOUND SCAN APPEARANCE OF RENAL PARENCHYMA AND KIDNEY LENGTH WITH SERUM CREATININE LEVELS IN CHRONIC KIDNEY DISEASE OF UNKNOWN ORIGIN Kaviratna M1, Wijesinghe KPS1, Wijebandara RJKS1, Attapathu NC1, Dharmaratne SD2, Wijeratna KHMAB1 1Base Hospital Mahiyanganaya; 2Department of Community Medicine, Faculty of Medicine, University of Peradniya. madurika.kaviratna@gmail.com

Mid Day Snack Break Presentation of Research Findings ENDEMIC CHRONIC KIDNEY DISEASE OF UNKNOWN AETIOLOGY OF SRI LANKA: HISTOPATHOLOGICAL CHANGES IN KIDNEYS OF ASYMPTOMATIC PATIENTS Wijetunge S1, Ratnatunga N2 , Ratnatunga C2 , Abeysekera DTDJ 3, Wazil AWM3, Selvarajah M4 Departments of 1 Pathology and 2 Microbiology, Faculty of Medicine, University of Peradeniya; 3Nephrology and Transplant Unit, Teaching Hospital Kandy; 4 Nephrology Unit, General Hospital Anuradhapura. suwijetunge@gmail.com

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SLMA­Research Symposium on CKDu

POTENTIAL OCCURRENCE OF CALCIUM ARSENATE DEPOSITIONS IN CKDu AFFECTED KIDNEYS Samarasinghe UDS1, Dahanayake KS2 1Base Hospital Karawanella; 2District General Hospital Moneragala. dhananjaya.samarasinghe@yahoo.com

ARSENIC (As) RELATED SKIN MANIFESTATIONS IN PATIENTS WITH CHRONIC KIDNEY DISEASE OF UNKNOWN AETIOLOGY (CKDu) IN THE NORTH CENTRAL PROVINCE OF SRI LANKA Dassanayake RT, Hulangamuwa S, Abeysekara DTDJ. rajeewa_d@yahoo.com

SPECIAL KIND OF PIGMENTATION IN PALMS AND SOLES OF INHABITANTS OF PADAVI SRI PURA­A CKDu ENDEMIC REGION IN SRI LANKA. Wijewardane KMRC1, Dahanayake KS2, Mahamithawa AMP3, Rajakaruna KDLMP4, Samarasinghe UDS5 1Divisional Hospital, Padavi Sripura; 2DGH‐Monaragala; 3Faculty of Medicine, Rajarata University; 4Base Hospital, Dambulla, 5Base Hospital Karawanella. mahamithawa2000@yahoo.com

TOXIC LEVELS OF ARSENIC IN HAIR AND URINE SAMPLES OF PATIENTS OF CHRONIC KIDNEY DISEASE OF UNKNOWN AETIOLOGY(CKDU) IN SRI LANKA. Jayasumana MACS1, Dahanayake KS 2, Paranagama PA3, Amarasinghe MD 3, Rajakaruna KDLMP4, Wijewardena KMRC 5. Mahamithawa AMP1, Samarasinghe UDS6, Fonseka SI3, Senanayake VK3,Wijekoon DVK7 1Faculty of Medicine, Rajarata University; 2DGH, Monaragala; 3Faculty of Science, University of Kelaniya; 4Base Hospital, Dambulla; 5Divisional Hospital, Padavi Sripura; 6 Base Hospital, Karawanalla; 7Water Resources Board, Colombo 7. jayasumanalk@yahoo.com

PRESENCE OF HIGH LEVELS OF ARSENIC IN INTERNAL ORGANS OF DECEASED PATIENTS WITH CHRONIC KIDNEY DISEASE OF UNKNOWN AETIOLOGY (CKDu): THREE CASE REPORTS. Dahanayake KS1,Wijewardahana KMRC2,Jayasumana MACS,3Paranagama P4 1District General Hospital Monaragala; 2Divisional Hospital Padavi Sri Pura; 3Faculty of Medicine, Rajarata University; 4University of Kelaniya. ksdahanayake@gmail.com

GRAPHITE FURNACE BASED (GFAAS) METHOD IS MORE APPROPRIATE THAN HYDRIDE GENERATOR BASED (HGAAS) METHOD IN USING ATOMIC ABSORPTION SPECTROMETRY TO DETECT ARSENIC IN HARDWATER COLLECTED FROM AQUIFERS OF CKDu ENDEMIC AREAS IN SRI LANKA. Paranagama PA, Fonseka SI, Jayalath K, Amarasinghe MD Faculty of Science, University of Kelaniya. priyani@kln.ac.lk

HARDNESS AND PRESENCE OF ARSENIC IN AQUIFERS OF SELECTED CKDu PREVALENT AND OTHER AREAS IN SRI LANKA Fonseka SI 1, Jayalath K1, Amarasinghe M1, Mahamithawa AMP2, Senanayake VK1, Paranagama PA1 1Faculthy of Science, University of Kelaniya; 2Department of Pharmacology, Faculty of Medicine, Rajarata University. siiresh@gmail.com

PRELIMINARY INVESTIGATIONS ON PRESENCE OF ARSENIC IN SOILS AND PLANTS FROM TWO CKDu PREVALENT AREAS IN SRI LANKA Amarasinghe MD1, Fonseka SI1, Jayalath K1, Senanayake VK1, Paranagama PA1, Jayasumana MACS2 4

SLMA­Research Symposium on CKDu 1Faculth of Science, University of Kelaniya; 2Department of Pharmacology,

Faculty of Medicine, Rajarata University. mala@kln.ac.lk EFFECT OF CONCENTRATED WATER FROM RESERVOIRS OF HIGH PREVALENCE AREA FOR CKD OF UNKNOWN ORIGIN IN SRI LANKA ON MICE Jayasekera JMKB1, Dissananyake DM1, Ratnayake P2, Wickramasinghe W3, Radella YA4, Palugaswewa WB5 1Department of Pathology, Faculty of Medicine, University of Peradeniya; 2Srimavo Bandaranayake Specialized Children Hospital, Peradeniya; 3National Environmental Toxicology Laboratories, University of Queensland, Queensland, 4Department of Medical Laboratory Science, Faculty of Allied Health Sciences, University of Peradeniya; 5Department of Irrigation, Anuradhapura. dhammika62@gmail.com

THE SHORT TERM EFFECT OF CYANOBACTERIALTOXIN EXTRACTS ON MICE KIDNEY Dissananyake DM1, Jayasekera JMKB1, Ratnayake P2, Wickramasinghe W3, Radella YA4, Shihana F1 1Department of Pathology, Faculty of Medicine, University of Peradeniya; 2Srimavo Bandaranayake Specialized Children Hospital, Peradeniya; 3National Environmental Toxicology Laboratory, University of Queensland, Queensland; 4Department of Medical Laboratory Science, Faculty of Allied Health Sciences, University of Peradeniya. dhammika62@gmail.com

INCREASING NUMBER OF CASES OF UROLITHIASIS OF UNKNOWN ORIGIN IN THANAMALWILA AND LUNUGAMVEHERA: COULD IT BE DUE TO THE SAME AETIOLOGY OF CKDu Wjewardane SS1, Weerasekara WJKLM2 , Rathnayake RMD 1Divisional Hospital, Lunugamvehera; 2Medical Office of Health Office, Thanamalwila; 3District General Hospital, Moneragala. shamindisw@gmail.com

SOCIOECONOMIC IMPACT OF CKDu: A PRELIMINARY ANALYSIS Liyanage C1, Jayathilake R1, de Silva A1, Kasthuriratne A2, Jayasinghe S 1University of Colombo1 and University of Kelaniya 2. sarojoffice@yahoo.com

PREVALENCE OF MAJOR DEPRESSIVE EPISODE AMONG PRE­DIALYSIS CHRONIC KIDNEY DISEASE PATIENTS Sumanathissa M1, De Silva V2, Hanwella R1 1Teaching Hospital, Kegalle; 3University of Colombo. msthissa@yahoo.com

PROVISION OF SAFE DRINKING WATER AS A STRATEGY FOR MITIGATION OF CHRONIC KIDNEY DISEASE IN SRI LANKA Ferdinando DNJ. National Water Supply and Drainage Board. nihalferdi@hotmail.com

01.45 pm

02.15 pm 02.45 pm

Getting to the aetiology of CKDu ­ A forensic pathologists perspective Dr. Ananda Samarasekara Judicial Medical Officer, Colombo Discussion Close

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SLMA­Research Symposium on CKDu

ABSTRACTS

EPIDEMIOLOGY OF CKDu

Dissanayake DM1, Jayasekera JMKB1, Ranasinghe AV2 1Department of Pathology, Faculty of Medicine, University of Peradeniya, Sri Lanka; 2Health Department, North Central Province, Anuradhapura. Sri Lanka. dhammika62@gmail.com

Introduction: Chronic Kidney Disease (CKD) is an emerging health problem all over the world. CKD of unknown aetiology reported in Sri Lanka shows similarities with that of Balkan endemic nephropathy and even more similarities with nephropathy described in Banana and sugarcane farmers of Nikaragua & Elsalvador.

Aim of the present study is to investigate the epidemiology of CKDu and to study pattern over the last 30 years.

Method & material: The information on the emergence of the disease was traced back to 32 years (1980‐ 2012) using statistics on live discharges and deaths due to different disease entities of G.H.Anuradhapura.

Monthly statistics from 1999‐2008 was collected and analyzed to study seasonal variation. Other demographic characteristics & stage of presentation was collected from community renal clinics & nephrology clinic of Anuradhapura.

Results: The male:female ratio was 2.4 :1 with mean age of 54.7 yrs. 90% of the patients were farmers.

92% had shallow wells as the source of drinking water. 29% of the patients show family clustering with no evidence of any Mendelian inheritance. Hospital based statistics showed an increasing trend in the number of patients with diseases of the genitor urinary system from 1980. CKDu appeared in early nineties for the first time and then the incidence of the disease amplified gradually reaching highest in 2002. The incidence of the disease has been stable but at a relatively lower level thereafter. Alcoholic liver disease reported to the G.H.Anuradhapura during the same time frame showed a similar distribution. A seasonal variation in mortality rate with peaks in November‐January was noted.

Conclusion: The reason for the male preponderance and lower age at diagnosis could be due to exposure of males to the toxins, more dehydration and exertion in males or sex linked gene like G6PD deficiency.

Familial occurrence of CKDu could be due to exposure of the members of the family to the same aetiological agent than genetic factor. Similarity in the variations of incidence over time in CKD‐U, alcoholic liver disease indicates the possibility of common aetiology. Seasonal variation in mortality in last few months of the year indicates the possibility of acute on chronic interstitial nephritis & needs further investigation

A STUDY OF GEOGRAPHICAL DISTRIBUTION OF CHRONIC KIDNEY DISEASE OF UNKNOWN ORIGIN IN SRI LANKA Dissanayake DM1 , Jayasekera JMKB 1 , Adhikari SB 2 , Bandara P3 1Department of Pathology, Faculty of Medicine, University of Peradeniya; 2 Mahaweli Development Authority, Kotmale; 3 Health Department, North Central Province, Anuradhapura. dhammika62@gmail.com

Introduction: An alarmingly high incidence of a new form of chronic kidney disease of unknown aetiology (CKDu) was noticed in some parts of Sri Lanka. Histopathological studies revealed a tubulo interstitial nephritis which is suggestive of a toxic aetiology. Similar endemic nephropathy described in Balken region shows relationship to Danubi river.

Objective: Aim of the present study is to investigate the geographical distribution of CKD‐U using modern GPS and GIS mapping.

Method & material: Information of 11630 patients were collected and used for GIS mapping using AR 9.2 software and GPS mapping.

Results: Results indicated five high prevalent areas in the region namely Medawachchiya, Padaviya, Girandurukotte, Medirigiriya and Nikawewa (identified 20, 18, 12, 8 & 5 yrs ago). Low prevalence of the disease was noted in communities who consume water from natural springs for drinking. In all five areas the distribution is related to stagnant irrigated water. GPS mapping showed most of the cases are located below the level of some reservoirs and some are related to the irrigation canals.

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SLMA­Research Symposium on CKDu All five regions affected with the CKD‐U encompass irrigation systems comprising of reservoirs used mainly for agricultural purposes. However, the villagers consume water from shallow wells and water levels of these shallow wells are proportionate to the water levels of the canals indicating that the ground water table is recharged from irrigation canals & reservoirs. Observations of the study reveals that the exposure to the aetiological agent remains unchanged and new disease foci are reported to be emerging.

Conclusion: Epidemiological data on geographical distribution infers that while older foci are persisting, there is an emergence of new foci of CKD‐U with the time. The presence of the affected villages located below the reservoirs and canals indicated the possibility of irrigated water draining to shallow wells which is the source of drinking water.

CHRONIC KIDNEY DISEASE OF UNCERTAIN AETIOLOGY PREVALENT IN SRI LANKA

Athuruliya NTC, Abeysekara TDJ, Amerasinghe PH, Kumarasiri R, Bandara P, Karunarathne U Department of Clinical Pharmacology, Faculty of Medicine, University of Peradeniya; Nephrology Unit, Teaching Hospital Kandy; International Water Management Institute, Colombo; Department of Community Medicine, University of Peradeniya; Ministry of Health, Colombo. ckdeupdnsl@yahoo.com

Background: Chronic kidney disease of uncertain etiology is prevalent in some provinces of Sri Lanka.

Objectives: To analysis the prevalence of proteinuric CKD of uncertain aetiology in some areas of North Central province(NCP), Central province(CP), Eastern province(EP), Southern province(SP) and Uva province(UP).

Method: Subjects were selected using the random cluster sampling method and those older than 19 years of age were screened for persistent dipstick proteinuria, diabetes mellitus, hypertension and other aetiologies of CKD. Results: Area NCP UP EP SP CP No of subjects 2600 913 2024 2844 709 Uncertain aetiology 84% 81% 72% 9% 2.9% Known aetiology 16% 19% 28% 91% 97.1% Conclusion: Protienuric CKD of uncertain aetiology is prevalent in some areas in NCP, UP and EP.

EPIDEMIOLOGICAL RISK FACTORS OF CHRONIC KIDNEY DISEASE OF UNKNOWN AETIOLOGY: A HOSPITAL BASED CASE CONTROL STUDY Chandrakumara WAJC1, Jankan N5, Marystella M3, Perera K4, Pushpakumara PHGJ2, Ruparathne AA1, Rupasinghe S2, Thilakaratne YGRKK1 1Department of Paediatrics; 2Department of Psychiatry, Faculty of Medicine & Allied Sciences, Rajarata University; 3Postgraduate Institute of Science; 4Department of Engineering Mathematics, Faculty of Engineering, University of Peradeniya; 5Epidemiolgy Unit. janakatechno@yahoo.com

Background: Chronic Kidney Disease of Unknown aetiology (CKDu) is a significant health problem in Sri Lanka. The geographical distribution of CKDu is mainly in the North Central Province (NCP) and to a lesser extent in North Western and Uva provinces. There are many theories on the culprit of CKDu including hard water, agrochemicals, fluoride content in water, use of aluminum utensils, snake bite, ayurvedic treatment and dietary Cadmium. Yet the exact cause for the CKDu still remains elusive.

Objective: Assessment of the epidemiological risk factors of the CKDu in NCP. Methods: 140 cases (70 males and 70 females) treated at Teaching Hospital Anuradhapura, were randomly drawn from National CKD registry. 140 sex matched controls with normal serum Creatinine level were selected from the same hospital. Data was collection was done using an interviewer administered questionnaire. Data was statistically analysed using R software and the odds ratios were calculated.

Results: The incidence of CKDu was increased in people whose occupation is agriculture for the longest part of life compared with those whose occupation for the longest part of life non‐agriculture related (Odds ratio [OR] 3.750, 95% confidence interval [CI] 2.160 to 6.509). Well water as the main source of drinking water for the last five years was a significant risk factor for CKDu (OR 3.773, CI 2.112 to 6.600).

Risk of CKDu was significantly higher among people who consumed illicit liquor (OR 5.667, CI 2.379 to 13.497) and among people who consumed tobacco/lime (OR 2.211, CI 1.286 to 3.800). People whose monthly income is less than Rs 10000/‐ had an increased risk of CKDu (OR 15, CI 5.452 to 41.271). Risk of CKDu did not differ significantly by smoking, alcohol intake, exposure to agrochemicals or snake bite.

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SLMA­Research Symposium on CKDu Conclusions: Results suggest that farmers, people whose source of drinking water is well, people who consume tobacco/lime and illicit liquor and people who have a monthly income of less than Rs 10000 are at an increased risk of CKDu.

OCCUPATIONAL RISK FACTORS & RISK INDEX FOR CHRONIC KIDNEY DISEASE OF UNKNOWN AETIOLOGY IN SRI LANKA

Jayasekara JMKB1, Dissanayake1 DM, Gunaratna MDN2, Thilakarathna S3 1Department of Pathology, Faculty of Medicine, University of Peradeniya; 2Department of Mathematics, Faculty of Engineering, University of Moratuwa; 3 Department of Medical Laboratory Sciences, Faculty of Allied Health Sciences, University of Peradeniya. dhammika62@gmail.com

Introduction: Health professionals in Sri Lanka have noticed worryingly high incidence of a new form of chronic kidney disease of unknown aetiology (CKD‐U) in North Central Region (NCR) of the Island. The epidemiology of the disease shows that most of the patients are paddy farmers.

Method & material: Three hundred and fifteen CKD‐U (315) patients (cases) and 321 healthy individuals were randomly selected and potential agricultural risk factors and other information were collected. The relative risk of each factor was compared in terms of Odds ratios (ORs), 95% CI and multiple linear logistic models were applied to calculate the risk index.

Results: Involvement in paddy farming activities (OR=1.945,95%CI 1.256‐3.010), usage of agro‐ chemicals (OR=2.034,95%CI,1.297‐3.190), low preventive measures against agro‐ chemicals(OR=2.845,95%CI 1.788‐4.527), high operated paddy extent(>1 hectare) without exchange of labour (OR=4.734,95%CI 2.586‐8.665) were identified as significant contributory risk factors (P<0.005).

Discussion: Being a male (OR=1.947, 95%CI 1.415‐2.927) and age > 60 years (OR=4.884, 95%CI 3.04‐ 7.415) (P<0.005) were identified as other contributory factors. Above identified risk factors were considered for index calculation. According to the multiple linear regressions logistic models, the maximum and minimum risks were calculated as 1.0 and 0.0 respectively. The males with age is greater than 60, low exchange of labour and low protective measures against agrochemicals had the highest risk of getting CKDu disregarding cultivating area (p=0.87).

Conclusion: Male farmers over 60yrs is at a high risk, probably due to long term exposure to aetiological agents (risk factors). Risk index will be useful in implementing preventive strategies and to identify individual risk of each person in particular areas.

RISK FACTORS ASSOCIATED WITH CHRONIC KIDNEY DISEASE OF UNKNOWN AETIOLOGY

Wanigasuriya KP1, Peiris‐John RJ2,3, Wickremasinghe R4 1Department of Medicine, University of Sri Jayewardenepura, Nugegoda; 2Department of Physiology, University of Sri Jayewardenepura, Nugegoda; 3Section of Epidemiology and Biostatistics, University of Auckland, Auckland, New Zealand; 4Department of Public health, University of Kelaniya, Ragama, kamaniw@sltnet.lk

1. CKDu in this region mainly affects males who are involved in paddy farming raising the possibility of agrochemical exposure as a cause for CKDu. A descriptive cross‐sectional study was carried out to determine if there is an association between CRF and low‐level organophosphate pesticide exposure.

There was greater inhibition of acetylcholinesterase among patients with CKDu compared with patients with chronic renal dysfunction from a non‐agricultural area.

2. In a case controlled study, 183 patients with CRF of unknown aetiology attending TH Anuradhapura were compared with a control group. In a multivariate logistic regression analysis significant predictors of CRF of unknown aetiology included, having a family member with a history of chronic renal dysfunction, a history of having taken ayurvedic treatment and having had a snake bite in the past. A family member with a history with renal dysfunction suggests a genetic aetiology of the disease but it is likely that the disease is triggered by an environmental factor in those genetically predisposed.

3. Risk factors for microalbuminuria (MA) were evaluated in of 425 females and 461 males CKDu. The prevalence of MA was 6.3% in females and 8.6% in males. In the binary logistic regression analysis the significant predictors of microalbuminuria included, age, history of smoking, being under treatment for hypertension and drinking well water in the fields. Subjects who drank well water in the field were approximately two‐and‐a‐half times more likely to have microalbuminuria as compared to those who do not do so.

4. Ochratoxin A (OA) levels were tested in 98 food samples, consumed by people in the NCP. The results indicate that OA is a natural contaminant of cereals and pulses cultivated in these areas, but the levels detected were below the statutory maximal limit.

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SLMA­Research Symposium on CKDu URINARY β2­MICROGLOBULIN VARIATION BETWEEN CHRONIC KIDNEY DISEASE PATIENTS OF UNKNOWN AETIOLOGY AND NON AFFECTED CONTROLS 1, Siriwardhana EARIE Perera PAJ1, Sivakanesan R2, Abeysekara T3 1Department of Biochemistry, Faculty of Medicine and Allied Sciences, Rajarata University; 2Department of Biochemistry, Faculty of Medicine, University of Peradeniya; 3Department of Pharmacology, Faculty of Medicine, University of Peradeniya. rangaedirisinghe@yahoo.co.uk

Introduction: Chronic kidney disease of unknown aetiology (CKDu) prevails in the North Central Province (NCP), parts of North Western and Uva provinces of Sri Lanka. Owing to the indistinct symptoms in early stages the disease remains undiagnosed to the end stage, at times, even requiring renal replacement therapy. Consequently early diagnosis becomes vital.

Objective: This study was initiated to investigate the variation in urinary β2‐microglobulin (β2M) levels of CKDu patients (Test) and to use it as a marker for early diagnosis.

Materials and Method: CKDu patients, males and females, were selected at random from Medawachchiya Divisional Secretariat formed the test group (n=30). Age and sex matched normal individuals from the same division (Control M, n=30) and from a different area, Ja – Ela (Control J, n=30) were used as controls. Spot urine samples from all subjects were analyzed in duplicate using an ELISA test kit.

Results: The mean urinary β2M level in CKDu patients (1.24 ± 0.71 µg/mL) was significantly higher (P<0.05) than that of control groups M (0.16 ± 0.05 µg/mL) and J (0.17 ± 0.05 µg/mL). Mean urinary β2M of males of the test group was significantly higher (p< 0.05) than that of the males of control groups.

Similar results were seen among the females.

Conclusion: Normal subjects from the same environment as CKDu patients did not show high urinary β2M levels. Among the CKDu patients, no significant difference was seen between the males and females in their urinary β2M levels.

Financial support from University Grants Commission and Rajarata University is acknowledged.

RENAL TUBULAR FUNCTIONS OF FARMERS OF HIGH PREVALENCE AREA FOR CKDu

Dissananayake DM, Jayasekera JMKB, Wimalasiri R, Ratnayake I, Dissanayake S Department of Pathology, Faculty of Medicine, University of Peradeniya. dhammika62@gmail.com

Introduction: The CKDu is common among the farmers of the North central region & mortality shows a seasonal variation of more cases with intense farming. The risk of developing CKDu is more with agricultural practices that show higher degree of exertion in farmland activities. Research on aetiological agents failed to identify a likely environmental toxin in toxic levels. The dehydration due to sweating in dry weather can have effects on the renal tubular functions.

Objective: The aim of this study is to assess the dehydration and its effects on renal functions of farmers from high prevalence area with a matched control group.

Method & material: Urine electrolytes & osmolality was determined in cases & controls before & after routine work & fluid balance was measured for the said period. Serum creatinine & electrolytes were determined from blood sample collected after routine work. The same test was repeated after provision of natural spring water to the test group for a period of two weeks.

Results: The results showed that the farmers have significantly low urine outputs, Na+, K+, Cl‐ & osmolality than controls before (p<0.05) & after work (p<0.05). Farmers with lowest values for Na+, K+, Cl‐, & osmolality showed significant improvement of these parameters after intervention (p<0.05).

Conclusion: The study shows possibility of a distal tubular dysfunction in farmers which showed some improvement with natural spring water. Either the intake of natural spring water or improved water intake due to better water quality may have improved the renal tubular defect. Using urine electrolytes & osmolality to detect early cases at a reversible stage of renal damage need further investigation.

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SLMA­Research Symposium on CKDu CORRELATION BETWEEN THE ULTRA SOUND SCAN APPEARANCE OF RENAL PARENCHYMA AND KIDNEY LENGTH WITH SERUM CREATININE LEVELS IN CHRONIC KIDNEY DISEASE OF UNKNOWN ORIGIN Kaviratna M1, Wijesinghe KPS1, Wijebandara RJKS1, Attapathu NC1, Dharmaratne SD2, Wijeratna KHMAB3 madurika.kaviratna@gmail.com Kaviratna M1, Wijesinghe KPS1, Wijebandara RJKS1, Attapathu NC1, Dharmaratne SD2, Wijeratna KHMAB1 1Base Hospital, Mahiyanganaya; 2Department of Community Medicine, Faculty of Medicine, University of Peradeniya. madurika.kaviratna@gmail.com

Introduction: Chronic kidney disease of unknown origin (CKDu) is a major noncommunicable disease prevalent in certain provinces of Sri lanka. Serum creatinine is used to monitor the disease progress. In addition, Ultra sound scan (USS) is done to detect the renal parenchymal changes and to rule out the possibility of an obstruction.

Aim: To assess the correlation between the echo pattern of renal parenchyma and the kidney lengths with serum creatinine level in diagnosed CKDu patients.

Materials and Method: Study composed of 122 diagnosed CKDu patients, 95 males and 27 females, aged 29 years to 85 years from the Base Hospital, Mahiyanganaya with their serum creatinine levels and USS reports. The cases were selected randomly. Kidney bipolar lengths were measured and echo pattern of renal parenchyma was graded from one to three by the Consultant Radiologist. Normal‐ 1, Slightly increased echo pattern‐ 2 and Increased echo pattern‐ 3.

Results and Discussion: The mean right kidney length is 7.99cm (SD= 1.04) and mean left kidney length is 8.01cm (SD= 1.07). Correlation coefficient between serum creatinine and right kidney length was ‐ 0.470 and was statistically significant at 0.01 levels. Correlation coefficient between serum creatinine and left kidney length was ‐0.405 and was statistically significant at 0.01 levels. Correlation coefficient between serum creatinine and renal parenchymal echo pattern was 0.428 and was statistically significant at 0.01 levels.

Conclusion: Results show that kidney length reduces and renal parenchymal echogenicity increases with the increase of serum creatinine levels in CKDu.

ENDEMIC CHRONIC KIDNEY DISEASE OF UNKNOWN AETIOLOGY OF SRI LANKA: HISTOPATHOLOGICAL CHANGES IN KIDNEYS OF ASYMPTOMATIC PATIENTS Wijetunge S1, Ratnatunga N2 , Ratnatunga C2 , Abeysekera DTDJ 3, Wazil AWM3, Selvarajah M4 Departments of 1 Pathology and 2 Microbiology, Faculty of Medicine, University of Peradeniya; 3Nephrology and Transplant Unit, Teaching Hospital Kandy; 4Nephrology Unit, General Hospital Anuradhapura. suwijetunge@gmail.com

Background: A chronic kidney disease of unknown aetiology (CKDu) is endemic in the north central regions of Sri Lanka.

Objective: to analyse the histopathological features of kidneys in asymptomatic patients with CKDu Methods: The study consisted of 234 asymptomatic individuals who have been detected by screening programmes and undergone renal biopsy during 2004 – 2011 period. Pathologic changes were categorized into 5 groups: 1, interstitial fibrosis +/‐ subtle tubular atrophy is the only change; 2, glomerulosclerosis with group 1 changes; 3, interstitial fibrosis, tubular atrophy and interstitial inflammation +/‐ glomerulosclerosis, 4, the predominant change is interstitial inflammation; 5, severe interstitial fibrosis with extensive glomerulosclerosis. Cases were divided into five clinical stages based on the glomerular filtration rate (GFR).

Results: There were 204/234 (87.2%) with interstitial renal disease. There were 65, 21, 115, 2 and 1 cases in groups 1 to 5 respectively. The mean GFR of groups 1 to 3 are 113.7±43.2, 86.8±54.8 and 52.2±30.4 respectively. The mean ages (in years) of groups 1 to 3 are 27± 12.9, 41.5 ± 14.2 and 41.7 ± 11.5 respectively. The number of cases in clinical stages 1 to 5 are 70 (34.3%), 28 (13.7%), 84 (41.2%), 22 (10.8%) and 0 (0%) respectively. The male to female ratio was 2.6:1.

Conclusions: The earliest pathological manifestation of endemic CKDu appears to be interstitial fibrosis and interstitial inflammation a late secondary phenomenon. When a significant reduction in GFR occur interstitial fibrosis and inflammation, tubular atrophy and glomerulosclerosis have set in. Even among the asymptomatic patients majority have either clinical stage 3 or 4 disease, further highlighting the need for active surveillance.

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SLMA­Research Symposium on CKDu

POTENTIAL OCCURRENCE OF CALCIUM ARSENATE DEPOSITIONS IN CKDu AFFECTED KIDNEYS Samarasinghe UDS1, Dahanayake KS2 1Base Hospital Karawanella; 2District General Hospital Moneragala. dhananjaya.samarasinghe@yahoo.com

Introduction: Arsenic present in drinking water is the most recently proposed (hypothesis) potential causative agent of chronic kidney disease of unknown aetiology (CKDu) in North Central Province and adjacent areas of Sri Lanka. Aetiology however is hitherto not been explained. Formation of calcium arsenate, the most probable molecule that can be expected to affect kidney function in the presence of Ca2+ and As under aerobic conditions needs further investigations for confirmation on the mechanisms involved.

Objective: To determine calcium and/or arsenic depositions in renal tissues of deceased patients as arsenic in hardwater has been identified as a major risk factor for the disease.

Methodology: Autopsy samples received from two pathological post mortem studies done at Padavi Sri pura were used for investigations. Renal tissues were processed to produce ten slides from each kidney.

Von Kossa method and copper sulfate were used as stains to visualize calcium and arsenic respectively.

Results: Occurrence of interstitial fibrosis and tubular atrophy were the principal histopathological observations made with the renal tissues under study. Severe atrophy and loss of glomeruli with areas of glomerularsclerosis and glomerular collapse were also noted. Characteristic dark colour of silver depositions when stained with Von Kossa method, was observed specially in atroping glomeruli.

Characteristic Paris green colour was produced in comparable regions when stained with copper sulfate, indicating presence of arsenates.

Conclusion: Tubular interstitial nephritis associated with severe glomerular atrophy and loss of tissue is the identifiable histopathological changes in these samples. It is commonly observed in toxic nephropathies. This combination is not usually seen in CKD of pure diabetic and hypertensive origin.

Arsenates produce Paris green colour with copper sulfate. Possibility of occurrence of calcium arsenate deposits cannot be excluded as areas which stained for calcium were also stained for arsenate. Chronic arsenic toxicity is known to produce tubular interstitial nephritis hence detail study is needed to assess the effect of calcium arsenate deposits in CKDu etiology.

ARSENIC (As) RELATED SKIN MANIFESTATIONS IN PATIENTS WITH CHRONIC KIDNEY DISEASE OF UNKNOWN AETIOLOGY (CKDu) IN THE NORTH CENTRAL PROVINCE OF SRI LANKA Dassanayake RT, Hulangamuwa S, Abeysekara DTDJ rajeewa_d@yahoo.com

Introduction: A recent presentation has claimed a high prevalence of arsenic (As) related skin manifestations among CKDu patients. In 12 CKDu endemic villages, almost 45% patients were thought to have As related skin manifestations. The same investigators and others, in a website by the Environmental Law Alliance Worldwide (ELAW) claim that almost 80% show the same manifestations.

We studied the prevalence of As related skin manifestations in renal clinic patients with CKDu. Materials and methods: A sample was selected from patients attending renal clinic, teaching hospital Anuradhapura. All patients with CKDu, attending on a single clinic day, were consented, and all consenting patients were referred to a consultant dermatologist, specifically for assessment of As related skin changes.

Results: 54 patients were identified and all patients consented. Data sheets after consultant dermatologist assessment were returned by 30. Only one had possible As related skin changes, and is planned for further investigations.

Discussion: It appears that no dermatologist was involved in the previous study. The online photographs do not appear to show characteristic dermatological manifestations. However, details are not clear in these photographs.

Conclusion: As related skin changes are rare, if present at all, in CKDu patients in the North Central Province.

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SLMA­Research Symposium on CKDu

SPECIAL KIND OF PIGMENTATION IN PALMS AND SOLES OF INHABITANTS OF PADAVI SRI PURA­A CKDu ENDEMIC REGION IN SRI LANKA Wijewardane KMRC1, Dahanayake KS2, Mahamithawa AMP3, Rajakaruna KDLMP4 , Samarasinghe UDS5 1Divisional Hospital,Padavi Sripura; 2DGH‐Monaragala; 3Faculty of Medicine, Rajarata University; 4Base Hospital, Dambulla; 5Base hospital Karawanella. mahamithawa2000@yahoo.com

Introduction: Padavi Sripura is located 120 km north east to Anuradhapura, in Trincomalee District.

Chronic kidney disease of unknown aetiology (CKDu) is emerging as the major health problem in the area.

It has been identified as the leading cause for deaths during the last few years. Due to the asymptomatic nature of CKDu it is not being able to detect until it reaches the final stage.

Objective: To determine and compare the occurrence of bronze colour spotty pigmentation in palms and soles of CKDu affected and unaffected individuals in Padavi Sripura.

Methodology: A descriptive cross sectional study was conducted at the divisional hospital, Padavi Sripura in June 2011. Patients attending medical clinic and OPD were subjected to thorough dermatological examination. Palms and soles were cleaned properly prior to the examination. Fifty six diagnosed CKDu patients and 62 individuals who haven’t diagnosed as CKDu patients were subjected to examination.

Results: Diameter of patches of bronze colour pigmentations that seemed to be located in deep layers of the skin varied from 3 mm to 25 mm. Most of them were circular or elongated in shape. It was prominent when placed in non pressurized areas. Slight illumination was noticed from these patches under torch light. Occurrence of spotty pigmentation was observed in palms of 42 (75%) and 48 (85.7%) soles of the CKDu patients observed during the study. Pigmentation was also observed in palms of 22 (35.4%) and soles of 26(41.9%) of non CKDu individuals.

Conclusion: Occurence of bronze colour spotty pigmentation of palms and soles of CKDu patients is significant and such an abnormal pigmentation has not been reported or documented previously. This couldn’t be due to CKDu alone as it is present even in individuals who have not been diagnosed to suffer from CKDu. Further investigations are needed to consolidate the occurrence and pathophysiology of this abnormal pigmentation.

TOXIC LEVELS OF ARSENIC IN HAIR AND URINE SAMPLES OF PATIENTS OF CHRONIC KIDNEY DISEASE OF UNKNOWN AETIOLOGY(CKDU) IN SRI LANKA Jayasumana MACS1.Dahanayake KS 2, Paranagama PA3, Amarasinghe MD 3, Rajakaruna KDLMP4, Wijewardena KMRC 5,.Mahamithawa AMP1,Samarasinghe UDS6, Fonseka SI3, Senanayake VK3,Wijekoon DVK7 1Faculty of Medicine, Rajarata University; 2DGH, Monaragala; 3Faculty of Science, University of Kelaniya, 4Base Hospital, Dambulla; 5Divisional Hospital, Padavi Sripura; 6Base Hospital, Karawanalla; 7Water Resources Board, Colombo 7. jayasumanalk@yahoo.com

Introduction: Chronic Kidney Disease of unknown aetiology (CKDu) is the main cause of deaths in at least 10 divisional secretarial divisions in Anuradhapura, Polonnaruwa, Mulative, Vavuniya and Badulla Districts.

Objectives: To determine the level of arsenic(As) in hair and urine samples of CKDu patients in Padaviya (Anuradhapura district) Sripura (Trincomale district) and Welioya (Mulative district) divisional secretarial areas and compare that with the values obtaining from non CKDu individuals from same region.

Study design, setting and methods: Case control study has been conducted. Cases were already diagnosed CKDu patients. (n=100) Control group (n=102) were selected from those individuals from the study area who have not been diagnosed to have CKDu. Hair and urine samples were collected from both CKDu patients and controls. Chemical analysis for As was carried out using atomic absorption spectrometer with graphite furnace (EPA 7060A) at University of Kelaniya and Water resources board separately.

Results: Hair and urine samples of CKDu patients were detected to contain 0.72±0.20 mg kg‐1 to 8.75±1.06 mg kg‐1 and 18.6 ±5.8 µg L‐1 to 94.50 ±10.2 µg L‐1 arsenic respectively. In Control group, the respective values of arsenic were 0.12±0.08 mg kg‐1 to 4.35±0.92 mg kg‐1 and 9.0± 3.2 µg L‐1 to 44.35 ± 7.9 µg L‐1.

Conclusion: Toxic level for arsenic in hair is 1mg/kg and 35 µg/L for urine. Findings of the present study reveal that hair and urine samples of 59 (59%) and 64 (64%) of the CKDu patients and 35 (34.3%) and 29

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SLMA­Research Symposium on CKDu (28.4%) of the subjects in the control group respectively contained toxic levels of arsenic.. As there is no evidence for mass scale acute or sub acute arsenic toxicity in the region, chronic accumulation may be the source of arsenic in biological samples. If arsenic is presumed to play a role in the aetiology of the disease, toxic levels of arsenic in urine and hair samples from individuals of the control group indicate that being inhabitants of the same area, majority of the population has already bio‐accumulated arsenic, nevertheless to a lesser extent than those who have been diagnosed as CKDu patients. This also implies that these individuals are of high risk in acquiring CKDu in near future. Furthermore, as it is not a deliberate self poisoning, Medico‐legal referrals should be done for these cases for subsequent legal action.

PRESENCE OF HIGH LEVELS OF ARSENIC IN INTERNAL ORGANS OF DECEASED PATIENTS WITH CHRONIC KIDNEY DISEASE OF UNKNOWN AETIOLOGY (CKDu): THREE CASE REPORTS Dahanayake KS1,Wijewardahana KMRC2,Jayasumana MACS,3Paranagama P4 1District General Hospital Monaragala, 2Divisional Hospital Padavi Sri Pura, 3Faculty of Medicine, Rajarata University, 4University of Kelaniya. ksdahanayake@gmail.com

Introduction: Chronic kidney disease of uncertain aetiology (CKDu), prevalent in the main rice cultivation regions under ancient irrigation system in Sri Lanka, has reached epidemic proportions over last two decades and it is considered to be one of the most important emerging health crises in the country. Over 10,000 patients in the region are currently being treated for CKDu. Case reports: Biopsy‐proven three CKDu patients (two males), aged 59, 55 and 62 years, expired at Padavi Sripura divisional hospital, were subjected to pathological autopsy. There was no known history of arsenic poisoning or any past history of diabetes, hypertension etc. related to them. All the bodies were in a fresh state.

External examination: There were no specific skin lesions other than spotty pigmentations in palms and soles.

Internal Examination: Kidneys have shown marked granulation and contractility. Severe cortical loss was the cardinal feature. Kidney capsules were easily stripped off from the cortical surface, which was characteristically seen in both cases. A Significant diffuse, small, fluid filled vesiculation was observed in both thyroid glands. Areas of Grey coloured discoloration were noted in lower poles of the kidneys, liver, spleen and in the wall of the rectum.

Arsenic contents of the internal organs were investigated using Atomic Absorption Spectrometer. Higher levels of arsenic were detected in kidney (213.5 ± 37.1 µg kg ‐1 ‐ 275.3 ± 45.8 µg kg‐1), liver (264.9±45.8 µg kg‐1 ‐ 295.8 ± 40.1 µg kg‐1) thyroid (187.2±44.6 µg kg‐1 ‐234.1±35.3 µg kg‐1),Spleen (155.6±35.5 µg kg‐1 ‐ 173.6 ±33.91 µg kg‐1) and in rectum (87.2±14.6 µg kg‐1 ‐ 194.1±25.3 µg kg‐1).

Conclusion: Arsenic content in the internal organs of the three CKDu victims were ten to twenty times higher when compared with the previously observed normal values for south Asian population by Muzumder in 2000 (20‐100 µg/kg). Arsenic levels were high in the samples obtained from grey coloured areas when compared with the samples obtained from other areas in the same organ. CKDu is not a definitive diagnosis as its underlying cause is not known yet. Therefore, it is mandatory to subject deceased patients to complete medico‐legal autopsy including histopathological and toxicological investigations for the confirmation of observations made in the present study.

GRAPHITE FURNACE BASED (GFAAS) METHOD IS MORE APPROPRIATE THAN HYDRIDE GENERATOR BASED (HGAAS) METHOD IN USING ATOMIC ABSORPTION SPECTROMETRY TO DETECT ARSENIC IN HARDWATER COLLECTED FROM AQUIFERS OF CKDu ENDEMIC AREAS IN SRI LANKA Paranagama PA, Fonseka SI, Jayalath K, Amarasinghe MD Faculty of Science, University of Kelaniya priyani@kln.ac.lk

Introduction: In water, arsenic is commonly found in inorganic species such as arsenite (AsO33‐) and arsenate (AsO43‐), which mostly have harmful effects on life. According to previously published data on presence of trace metals in groundwater in CKDu‐ prevalent areas in Sri Lanka show that As‐content measured in groundwater is lower than the maximum permissible level, i.e. 10 ppb. Apart from spotted and diffused pigmentation in palms/soles, relatively high levels of As was detected in urine and hair

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SLMA­Research Symposium on CKDu samples of CKDu patients. These unconvincing evidence on sparing presence of As in groundwater therefore demanded further investigations.

Objective: To investigate the presence of arsenic in groundwater using Atomic Absorption Spectrometry with Hydride Generator (HGAAS) and Graphite Furnace (GFAAS) in CKDu prevalent areas of Sri Lanka and it’s potential link to the hardness of groundwater.

Study design, setting and methods: Water samples in triplicate were collected from CKDu endemic areas, both from ground and surface water sources as well as from non‐CKDu areas, as the controls. A total of 88 water samples were collected from above areas. Hardness (Ca2+ and Mg2+) of water were measured using EDTA tritration (EPA 130.2) and arsenic contents in water samples were detected using GFAAS (EPA 7060A) and HGAAS (EPA 1632) separately. Relationship between As content measured using GFAAS and hardness of all 88 water samples was statistically analyzed using linear regression and criterion for significance was set at p< 0.05 and analyses were performed using Minitab 2.5.

Results: Total hardness ranged from 150 – 815 mg L‐l and the results indicated that even within a smaller area, groundwater hardness varies considerably. Comparison of mean As contents detected with GFAAS and HGAAS reveals that the As content measured using the two methods is significantly different (P< 0.05) from each other, confirming that the As content measured using GFAAS shows a statistically significant increase to that measured with HGAAS. For example As content of a hardwater (410 mgL‐1) sample when measured with GFAAS and HGAAS was 40.23±1.67 µgL‐1 and 0.6±0.21 µgL‐1 respectively.

One of the reasons could be the hardwater in the CKDu‐endemic areas that is rich in Ca2+ can form thermodynamically stable hydrates, (Ca3(AsO4)2.xH2O, Ca4(OH)2(AsO4)2.4H2O, Ca5(AsO4)3.4H2O) which are not readily ionized in aqueous solutions. The relationship between total hardness and their arsenic content of groundwater samples tested shows a statistically significant (p<0.05) linear relationship among the two variables.

Conclusion: Atomic Absorption Spectrophotometry with Graphite Furnace (GFAAS) was proved to be more appropriate in measuring arsenic in hardwater and the results generated with GFAAS revealed a positive relationship between arsenic content and groundwater hardness.

HARDNESS AND PRESENCE OF ARSENIC IN AQUIFERS OF SELECTED CKDu PREVALENT AND OTHER AREAS IN SRI LANKA Fonseka SI 1, Jayalath K1, Amarasinghe M1, Mahamithawa AMP2, Senanayake VK1, Paranagama PA1 1Faculthy of Science, University of Kelaniya; 2Department of Pharmacology, Faculty of Medicine, Rajarata University. siiresh@gmail.com

Introduction: Presence of arsenic in drinking water is a serious public concern as it is a class I poison that affects human health. A frequent complaint of the inhabitants of chronic kidney disease of unknown aetiology (CKDu) endemic areas, who primarily are farmers, is the increasing hardness in dug wells and tube wells that receive water from aquifers.

Objective: To determine and compare the total hardness and arsenic content in aquifers in selected CKDu prevalent and non prevalent areas in Sri Lanka Study design, setting and methods: Groundwater samples were collected from dug wells in Padavi‐Sri Pura (n=36) and Polpithigama (n=17) areas where chronic kidney disease of unknown aetiology (CKDu) is prevalent as well as from Moneragala (n=38) and from Thanamalwila (n=19) where no CKDu patients present, nevertheless a significant number of patients with calculus kidney disease have been reported.

Besides, groundwater from Matale (n=11), and Pasgoda‐Deniyaya (n=10), where no CKDu incidences have been reported too were collected and all water samples were analyzed for total hardness and for arsenic by using the atomic absorption spectrometer with graphite furnace detector.

Results: Average hardness in Padavi‐Sripura (270+54 – 820+62 mg L‐1) and Polpithigama (90+8 – 615+47 mg L‐1) was greater than that of Moneragala (10+2 – 340+31 mg L‐1), Thanamalwila (170+8 ‐ 500+24 mg L‐1) and Matale (60+5 – 460+21 mg L‐1). Hardness of Pasgoda water was < 60+6 mg L‐1 and no arsenic was detected in it. The average contents of arsenic in groundwater varied among areas, i.e.

Padaviya (21.07+3.54 ‐ > 100.91+12.31 μg L‐1), Polipithigama (2.49+0.61 – 60.55+7.21 μg L‐1), Moneragala (2.14+0.84 – 52.47+6.71 μg L‐1), Matale (1.02+0.08 ‐37.1+4.4 µg L‐1) and Thanamalwila (39.37+5.21‐ >100.42+9.45 μg L‐1).

Conclusion: A strong positive (p<0.05) correlation was revealed between the arsenic content and groundwater hardness in CKDu prevalent areas when compared to the other areas that showed a weak correlation between the two variables. The present study also indicated that arsenic associated with elevated levels of hardness can reasonably be one of the potential causes of CKDu.

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SLMA­Research Symposium on CKDu

PRELIMINARY INVESTIGATIONS ON PRESENCE OF ARSENIC IN SOILS AND PLANTS FROM TWO CKDu PREVALENT AREAS IN SRI LANKA Amarasinghe MD1, Fonseka SI1, Jayalath K1, Senanayake VK1, Paranagama PA1, Jayasumana MACS2 1Faculth of Science, University of Kelaniya; 2Department of Pharmacology, Faculty of Medicine, Rajarata University. mala@kln.ac.lk

Introduction: In recent years a significant increase in patients of Chronic Kidney Disease of unknown aetiology (CKDu) has been observed in some parts of Sri Lanka, especially in North central province.

Arsenic is well recognized as an element of public concern due to its high toxicity and carcinogenic properties. The present study was carried out in relation to the hypothesis that presence of arsenic in the groundwater aquifers in CKDu prevalent areas is the potential cause of the disease.

Objective: To determining arsenic content in soil profiles and selected aquatic and terrestrial plants in Padaviya and Polpithigama/ Nikawewa areas with a view to understanding the vertical and horizontal (spatial) distribution of arsenic in the environment.

Study design, setting and methods: Soil samples were taken in triplicate at 1 foot depths down to 12 feet from paddy fields, homesteads and draw‐down areas of Padaviya reservoir using a spiral auger.

Samples of plant parts from common plants in the study areas were tested for presence of arsenic using the atomic absorption spectrometer after acid digestion.

Results: All surface soil samples contained total arsenic contents greater than those in the bottom horizons of the soil profiles. Surface layers of soil in paddy fields of Padaviya area were detected to have relatively high levels of arsenic (1.5 mg kg‐1) when compared to that of the deep layers (0.61 mg kg‐1). No arsenic was detected below 7 feet depth in Padaviya reservoir. Bark of Azadirachta indica (Kohomba) (> 100 µg L‐1) was found to accumulate the greatest amount of total As while it was observed that roots and leaves of Terminalia arjuna (115±2.4 µg L‐1 in bark, > 100 µg L‐1 in roots) too accumulate As more than the other tree species. Eichhornia crassipes (553.5± 2.4 µg L‐1), the aquatic floating plant and flowers of Nelumbo sp.(rooted aquatic plant) (1101± 10.2 µg L‐1) were found to contain excessive amounts of arsenic.

Conclusion: The results of the present study revealed that presence of arsenic in the soils and plants, particularly in the agricultural areas gradually decreases with depth, indicating that it is not present naturally in the bedrocks nevertheless has been introduced from the surface, most probably due to anthropogenic activities such as agrochemicals.

EFFECT OF CONCENTRATED WATER FROM RESERVOIRS OF HIGH PREVALENCE AREA FOR CKD OF UNKNOWN ORIGIN IN SRI LANKA ON MICE Jayasekera JMKB1, Dissananyake DM1, Ratnayake P2, Wickramasinghe W3, Radella YA4, Palugaswewa WB5 1Department of Pathology, Faculty of Medicine, University of Peradeniya; 2Srimavo Bandaranayake Specialized Children Hospital, Peradeniya; 3National Environmental Toxicology Laboratories, University of Queensland, Queensland; 4Department of Medical Laboratory Science, Faculty of Allied Health Sciences, University of Peradeniya; 5Department of Irrigation, Anuradhapura. dhammika62@gmail.com

Introduction: Epidemiology of CKDu shows distribution of patients around the some water reservoirs and most of them are farmers. Low prevalence of the disease among villagers who use water from natural springs was observed. Based on the hypothesis that water is the carrier of the CKD‐U causing agent, the potential effects of concentrated water of one of these reservoirs in the high prevalence area on the kidneys was studied by mouse bioassay.

Method& material: Water of Padaviya reservoir supplying water to a high prevalent area was concentrated fifteen times by evaporation, exposing to sunlight. The test group of mice (20) and control group (15) were fed with concentrated water and Kandy water respectively for 6 months and sacrificed to examine the histology of kidneys. Water samples were analyzed for Fluoride, Na + and K+ using ISE method, heavy metals using ICP/MS and for cyanobacterial toxins microcystin & cylindrospermopsin using LC/PDA and LC/MS/MS respectively.

Results: At the end of 6 months interstitial nephritis was detected in 45% test mice and 6.5% controls (p< 0.001). Concentrated water samples showed high concentrations of fluoride, Na+, K+ but not heavy metals. The cyanobacterial toxin analysis showed low levels (0.05 ug/L ) of deoxy‐cylindrospermopsin.

The analysis of concentrated water samples showed significantly high content of fluoride (2.25 mg/L), Sodium (225 mg/L) (p<0.05) than control samples. However, no increased levels of heavy metals were

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SLMA­Research Symposium on CKDu detected. The analysis showed presence of Deoxy cylindrospermopsin (1.28 ug/L) as the predominant isomer present over cylindrospermospsin which is unusual.

Conclusion: The results show the ability of water of this reservoir to induce interstitial nephritis that could be due to the high salinity, fluoride or due to DCYN. Although present in low levels, the possibility DCYN to induce interstitial nephritis needs to be investigated further as the epidemiological evidence is in favor of a cyanobacterial toxin. Long term effects and safe levels for DCYN in drinking water & the effect of high salinity & high fluoride content of water needs to be studied. The study shows the need of an alternative, water supply should be considered for these villages, irrespective of the source of contamination of the shallow groundwater supplies.

THE SHORT TERM EFFECT OF CYANOBACTERIALTOXIN EXTRACTS ON MICE KIDNEY

Dissananyake DM1, Jayasekera JMKB1, Ratnayake P2, Wickramasinghe W3, Radella YA4, Shihana F1 1Department of Pathology, Faculty of Medicine, University of Peradeniya; 2Srimavo Bandaranayake Specialized Children Hospital, Peradeniya; 3National Environmental Toxicology Laboratory, University of Queensland, Queensland; 4Department of Medical Laboratory Science, Faculty of Allied Health Sciences, University of Peradeniya. dhammika62@gmail.com

The epidemiology of the chronic kidney disease of unknown origin (CKDu) in Sri Lanka shows distribution of patients around the water reservoirs. Similarity in the variations of incidence over time in CKD‐U and alcoholic liver disease in the North Central Region indicates the possibility of a toxin with hepatotoxic & nephrotoxic effects. Histopathology of the renal disease shows evidence of a tubulointerstial nephritis indicating a possibility of toxic aetiology. Some cyanobacteria that exist in water reservoirs are capable of secreting toxins in certain adverse environmental conditions. Cyanobacterial toxins are known to have hepatotoxic, dermatotoxic, neurotoxic effects in humans and nephrotoxic effects in experimental animals.

The aim of the study is to find the short term effects of extracts of cyanobacteria isolated from the affected reservoirs and canals of the high prevalence area for CKD‐U on mice kidneys.

Diluted extracts of Lyngbia bloom,microcystis bloom and mixed bloom with predominant cylindrospermopsis, from Ulhitiya& Padaviya reservoirs and canals was fed to a group of 5, 7 &10 mice respectively for a week. Another 5 mice fed with diluted extracts of microcystis bloom for one week of followed by 2 weeks of normal water. Control group of mice (10) were given normal water for a week.

Diluted crude extracts were analyzed for cyanobacterial toxins using LC/PDA and LC/MS/MS respectively for microcystin & cylindrospermopsin.

Acute tubular necrosis (ATN) was detected in 5/5 mice fed with extracts of microcystis bloom that contained microcystin (65µg/l), Deoxy Cylindropspermopsin (DCYN) (2.1 µg/l) and Cylindropspermopsin (CYN) while 2/5 mice had ATN when this extract was followed by 2 weeks of normal water. One out of seven mice fed with Cylindrospermopsis bloom that contained DCYN (29.5 µg/l) and CYN (0.7 µg/l) had ATN. Six out of 10 mice fed on Lyngbia bloom containing CYN (1.7 µg/l) & DCYN (0.5 µg/l) had acute tubular necrosis. All control mice had normal tubules.

The results show the ability of the cyanobacterial extracts to induce ATN in mice in the given concentrations. The ability of the normal water to reverse the activity to a certain extent was seen when fed with normal water for 2 weeks. As DCYN was available in all extracts the ability of DCYN to induce tubular necrosis even at low concentrations need to be investigated.

INCREASING NUMBER OF CASES OF UROLITHIASIS OF UNKNOWN ORIGIN IN THANAMALWILA AND LUNUGAMVEHERA: COULD IT BE DUE TO THE SAME AETIOLOGY OF CKDu Wjewardane SS1, Weerasekara WJKLM2, Rathnayake RMD 1Divisional Hospital, Lunugamvehera; 2Medical Office of Health Office, Thanamalwila; 3District General Hospital, Moneragala. shamindisw@gmail.com

Introduction: Aetiology of Urolithiasis includes high dietary salt intake. It may be naturally occurring or due to anthropologic activity such as irrational agrochemical use, causing high salt content in drinking water sources. Hardness is associated with high ion content of water and there is a known positive co‐relation between hardness and arsenic content in water.

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SLMA­Research Symposium on CKDu Objectives: To study the increasing trend of urolithisis in Thanamalwila and Lunugamvehera areas.

Methodology: Descriptive study of incidence of urolithiasis has been performed by using IMMR based statistics of DH Thanamalwila and DH Lunugamvehera.. Multiple random sampling of drinking water sources from these areas was done. They were analyzed for hardness and arsenic content using atomic absorption spectrometer with graphite furnace detector.

Results: Increasing trend of cases of urolithiasis has been identified. Three, 07 and 15 cases have been reported from Thanamalwila in year 2009,2010 and 2011 respectively. Seven, 11,09,46 and 48 cases have been reported from Lunugamwehera from 2007 to 2011. Water samples from Lunugamwehera and Thanamalwila detected to contain 12+3– 352+34 mg L‐1 and 170+8 ‐ 500+24 mg L‐1 hardness respectively. Water hardness had shown a correlation with the arsenic levels. Arsenic levels were 2.14+0.84 – 52.47+6.71 μg L‐1 in Lunugamvehera and 39.37+5.21‐ >100.42+9.45 μg L‐1in Thanamalvila.

Conclusion: There is an increasing trend of urolithiasis and high hardness and arsenic content of drinking water sources. Further studies are needed to find out any possible link between urolithiasis, high water hardness and high arsenic content of the drinking water sources. In CKDu endemic areas it was found to be having very high hardness and high arsenic content in ground water. Though such a very high level of hardness and arsenic is not observed, possible role by those factors in urolithiasis cannot be excluded.

SOCIOECONOMIC IMPACT OF CKDu: A PRELIMINARY ANALYSIS Liyanage C1, Jayathilake R1, de Silva A1, Kasthuriratne A2, Jayasinghe S University of Colombo1 and Kelaniya2 sarojoffice@yahoo.com

Introduction: CKDu is estimated to affect 8 to 9% of adults in rural NCP, and led to NCP having the highest number of deaths due to CKD in the country. The present study describes the socio‐economic impact of the disorder in these areas.

Methods: Studies included a community‐based study to describe social impact and estimate costs at the household and community level, and a hospital‐based study to estimate costs to the health system. The community‐based study consisted of interviewing 200 patients with CKDu patients selected from registers.

Results: Most were poorly educated (12.5% had passed GCE O‐Level) and from poor socio‐economic group. 131 (65.5%) were males and 184 (92%) above 40 years. 157 (78.5%) found that CKDu reduced their contribution to household income. Costs of agriculture increased because of shortage of labour, costs of hiring workers and machinery. However, medical costs increased due to laboratory tests, medication and transport. Allowances from social service sector were meager and families were stigmatized as a result of the diagnosis.

Conclusions: CKDu is devastating rural communities in NCP. Its national impacts could include slower economic growth and depopulation of areas. The aetiology appears to be linked to water. Focus on bio‐ medical aetiologies and interventions to tackle it should work at least in parallel or perhaps take a back seat and instead the government should invest in a massive development effort to provide safe water and social protection. Irrespective of CKDu, safe water is immensely beneficial and rights of this voiceless group should be respected.

PREVALENCE OF MAJOR DEPRESSIVE EPISODE AMONG PRE­DIALYSIS CHRONIC KIDNEY DISEASE PATIENTS Sumanathissa M1, De Silva V2, Hanwella R2 1Teaching Hospital, Kegalle; 2University of Colombo. msthissa@yahoo.com

Objective: To ascertain the prevalence of depression among pre‐dialysis chronic kidney disease (CKD) patients.

Methods: A cross‐sectional study was carried out using the Structured Clinical Interview for DSM IV (SCID) to detect major depressive episode. Study was carried out in the nephrology out‐patient clinic at Teaching Hospital Anuradhapura. Every fifth patient with CKD diagnosed according to the K/DQOI of the National Kidney Foundation of USA criteria stages 2‐5 not undergoing dialysis was recruited. Primary

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SLMA­Research Symposium on CKDu outcome was major depressive episode diagnosed using the Structured Clinical Interview for DSM Disorders (SCID).

Results: Sample consisted of 140 patients. Eighty nine were male. The mean age was 57.9 years (SD=10.4). Only 41 (29.3%) were in paid employment. Percentages of patients in CKD stages2, 3, 4 and 5 were 2.9%, 19.6%, 51.4% and 25% respectively. Only three patients had diabetes. One hundred and five were on treatment for hypertension. Prevalence of major depressive episode was 27.9%. Among males prevalence was 27% (95% CI 17.6‐36.3) and among females, 29.4% (95% CI16.5‐42.4). Age, gender, income, employment states and education were not associated with depression. The only significant variable associated with depression was patient’s understanding of prognosis.

Conclusions: Prevalence of major depressive episode among pre‐dialysis CKD patients was 27.9%. Rate of depression diagnosed using structured clinical interview was lower than that reported when screening instruments were used. The only significant variable associated with depression was patient’s understanding of prognosis. Future studies should aim to identify risk factors for depression among patients with chronic kidney disease.

PROVISION OF SAFE DRINKING WATER AS A STRATEGY FOR MITIGATION OF CHRONIC KIDNEY DISEASE IN SRI LANKA Ferdinando DNJ National Water Supply and Drainage Board. nihalferdi@hotmail.com

Chronic Kidney Disease (CKD) which is prevalent in the North Central Province and certain other areas in Sri Lanka is considered a major health hazard. In these areas a considerable percentage of the population including young adults and children are affected. CKD has considerable socio economic impacts on the communities and it is even considered as a social stigma. Extensive research to find the root cause of the disease has been conducted by several local as well as foreign agencies. Several hypotheses attributing the disease to cadmium, pesticides, fluoride, arsenic, algal toxins etc. have been presented. Yet the real cause is not established.

The areas affected are largely paddy cultivation areas. These communities mainly use groundwater from dug wells and hand pumps for their consumption. In the entire affected area groundwater is found to be hard and contain fluoride exceeding the WHO standards. Areas in NCP where the water supply is surface water based, are free from CKD. Though the possible effect of hardness is not well established, it is medically accepted that water containing fluoride is extremely detrimental to kidney patients.

Considering the water source limitations and the quantum of investments needed, it is not practicable to supply all affected village areas with surface water based pipe borne water. An alternative approach which combines advanced water treatment technology with a simple transport mechanism using water tankers to supply community tanks, is proposed as an alternative viable solution. Implementation of such a strategy is considered effective in mitigating CKD in Sri Lanka.

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