1. Microbiological quality of water spinach grown in the Boeng Cheung Ek lake in Phnom Penh and in peri-urban aquatic systems in Hanoi Vuong Tuan Anh 1, Lise Tønner Klank 2, Phung Dac Cam 1, Anders Dalsgaard 2. 1 Division of Enteric Infection Diseases, National Institute of Hygiene and Epidemiology, Vietnam; 2 Department of Pathobiology, Royal Veterinary and Agricultural University, Denmark

2. Study objectives 1.To determine the numbers of thermotolerant coliforms, helminth eggs and protozoan parasites in water spinach grown in Boeng Cheung Ek (BCE) lake, Phnom Penh and at two peri-urban communes in Hanoi, including samples harvested with and without wastewater (ww) exposure 2.To assess the association between microbiological water quality and of water spinach

3. Sites and methodology l Sampling sites and schedule l Phnom Penh: 2 inlets, 1 outlet of BCE lake and the control site (a small pond); all sites sampled four times; with and without ww exposure of plants l Hanoi: one ww-irrigated site, one rainwater-irrigated site, 6 fields at each site sampled during wet and dry seasons (duplicate samples) l Number of sample collected: 68 plant samples in Phnom Penh; 216 plant samples in Hanoi

4. Sites and methodology l Parameters: thermotolerant coliforms, E. coli, helminth eggs (Ascaris lumbricoides, Trichuris trichiura, hookworm) and protozoa (Giardia intestinales, Cryptosporidium parvum and Cyclospora spp.) l Sample analysis: Cambodian Pasteur Institute and NIHE l Laboratory methods: MPN for thermotolerant coliforms and helminth egg enumeration according to WHO manual 1996, Immuno-flourescent microscopy to detect protozoan parasites

5. Phnom Penh results: Numbers of thermotolerant coliforms and E. coli in water spinach

6. Phnom Penh: protozoan parasites in water spinach (total 68 samples)

7. Phnom Penh: helminth eggs qualitative analysis Sampling location Hook- worm Ascaris spp. Trichuris spp. JICA 1 0/12 Trabek 1 0/121/120/12 Steung Chrov 2 1/122/120/12 Control0/81/80/8 1 inlet; 2 outlet

8. Phnom Penh: nos of thermotolerant coliforms in water spinach exposed versus non-exposed to ww during harvest

9. Correlation of microbiological quality of water and water spinach

10. Hanoi results: Thermotolerant coliforms in water spinach (log MPN/g)

11. Hanoi results: Seasonal occurrence of protozoan parasites in ww-irrigated and rainwater-irrigated water spinach (%) (total of 216 samples) in peri-urban Hanoi

12. Hanoi results: helminth egg qualitative analysis Sampling location Hook- worm Ascaris spp. Trichuris spp. WW-irrigated site – Wet season (n=48) 0/481/480/48 WW-irrigated site – Dry season (n=48) 0/482/480/48 Non-WW-irrigated site – Wet season (n=60) 0/60 Non-WW-irrigated site – Wet season (n=60) 0/60

13. Phnom Penh study – Conclusions Thermotolerant coliforms –Relatively higher levels (10 5 -10 6 /g) on water spinach Protozoan parasites –Giardia is most prevalent, particularly in plant samples collected at inlets Helminth eggs –Very low levels detected at all sampling sites

14. Hanoi study – Conclusions Thermotolerant coliforms –Relatively low levels on water spinach (<10 4 /g) –No apparent differences between seasons Protozoan parasites –Higher prevalences during dry season at WW-site –Giardia is most prevalent, particularly at WW-site –Cryptosporidium: high levels during dry season at WW- site –Cyclospora is less prevalent than Giardia and Cryptosporidium, but is present at both sites and during both seasons Helminth eggs –Very low levels detected at both sites, no apparent difference between seasons

15. Study completion and remaining work Data analysis PCR analysis for identification and characterization puruposes; and subtyping of protozoans Preparation and submission of manuscripts (1-2 MS)

16. Acknowledgements Staff of the Faculty of Fisheries, at the Royal University of Agriculture, in particular Chouk Borin, and at the Pasteur Institute in Phnom Penh for excellent technical assistance in sample and data collection The National Inst of Hygiene and Epidemiology (NIHE) for providing laboratory facilities and good laboratory support for sample analyses EU-INCO-DEV (PAPUSSA), DANIDA (grant no. 104.Dan.8.L ”Sanitary Aspects of Drinking Water and Wastewater Reuse in Vietnam”) and KVL for financial support