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Cryptosporidia and Giardia analytics and challenges Dr. Katrin Luden

Dr. K. Luden. Cryptosporidia and Giardia analytics and challenges Dr. Katrin Luden. Warsaw March 2009 1. Dr. K. Luden. C. parvum obligat intracellular parasites causing diarrhea Infections caused by oocysts (5 µm) containing sporozoites (infection dose 1-10 oocysts)

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Cryptosporidia and Giardia analytics and challenges Dr. Katrin Luden

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  1. Dr. K. Luden Cryptosporidia and Giardia analytics and challenges Dr. Katrin Luden Warsaw March 2009 1

  2. Dr. K. Luden C. parvum obligat intracellular parasites causing diarrhea Infections caused by oocysts (5 µm) containing sporozoites (infection dose 1-10 oocysts) transmitted by feacal oral route or contaminated water Reservoir: vertebrates e.g. cattle, horses, goats, sheep dogs cats, birds ... Incubation time 7-10 (1-12) days Excretion with feaces for several weeks possible So far no specific therapy available Preventive measures heating to 60°C for 30 min or boiling Cryptosporidium Warsaw March 2009 2

  3. Dr. K. Luden G. lamblia parasite of the small intestine causing diarrhea Infections caused by cysts (7x 12 µm) containing trophozoites transmitted by feacal oral route or contaminated water Reservoir: humans, vertebrates? Incubation time 3 - 25 (1-14) days Drug therapy available May persist for years asymptomatically Giardia Warsaw March 2009 3

  4. Dr. K. Luden Annex I Part C: Indicator Parameters Clostridium perfringens Note 2: This parameter need not be measured unless the water originates from or is influenced by surface water. In the event of non-compliance with this parametric value, the Member State concerned must investigate the supply to ensure that there is no potential danger to human health arising from the presence of pathogenic micro-organisms, e.g. cryptosporidium. Member States must include the results of all such investigations in the reports they must submit under Article 13(2). Drinking water directive 98/83/EC Warsaw March 2009 4

  5. Dr. K. Luden Annex I Part C: Indicator Parameters Clostridium perfringens This parameter need not be measured unless the water originates from or is influenced by surface water. In the event of non-compliance with this limit value, the competent authority arranges for investigation into the supply system to ensure that there is no potential danger to human health arising from the presence of pathogenic micro-organisms, e.g. cryptosporidium. The competent authority informs the Federal Ministry for Health via the highest Land authority. German drinking water ordinance Warsaw March 2009 5

  6. Dr. K. Luden Methods regularly applied in US and UK: US EPA 16223 and UK (DWI), similar to ISO 15553 Limit values and methods Warsaw March 2009 6

  7. Dr. K. Luden Water quality – Isolation and identification of Cryptosporidium oocysts and Giardia cysts from water Scope: no species identification, no determination of viability no determination of infectivity Principle: Concentration form water by filtration and elution followed by low speed centrifugation Purification by immunomagnetic separation (IMS) Detection by epifluorescence and differential interference contrast (DIC) microscopy ISO 15553 Warsaw March 2009 7

  8. Dr. K. Luden Sampling: 10 to 1.000 l of water – into transport containers (grab samples) or filtering in the fieldflow rate and pressure may be limited by the filter used Roughly 95-100 % of parasites are retained by the filter Recovery rate of the whole method varies widely (10-80 %) depending on the matrix Turbidity important (adhesion of oocysts/cysts to particles?) ISO 15553 Warsaw March 2009 8

  9. Dr. K. Luden Concentration Pump peristaltic regular Flow meter(oocysts/volume determination) Filter capsule Envirocheck, Filta Max Sampling site (source water, treated water, tap) Discharge Warsaw March 2009 9

  10. Dr. K. Luden Filter capsule is washed and elution buffer collected in centrifugation tubes Centrifugation Supernatant discarded Pellet -purification/IMS Concentration Warsaw March 2009 10

  11. Dr. K. Luden Filter capsule is washed and elution buffer collected in centrifugation tubes Centrifugation Supernatant discarded Pellet -purification/IMS Concentration Warsaw March 2009 11

  12. Dr. K. Luden Immunomagnetic separation Purification by IMS Paramagnetic bead Anti-cryptosporidium-antibodies Oocyst Y Y Complex separated from debris using magnets and washing Warsaw March 2009 12

  13. Dr. K. Luden IMS Warsaw March 2009 13

  14. Dr. K. Luden Immunomagnetic separation Y Purification by IMS/Detection Staining with FITC labelled antibodies Dissociation of the complex by acid-(heat) treatment Y Detection by microscopy Warsaw March 2009 14

  15. Dr. K. Luden Detection Epifluorescence microscopy and DIC (Differential interference contrast) Counterstaining with DAPI is recommended to detect nuclei of sporozites and trophozoites DIC detects internal morphological characteristics Cryptosporidium oocyst 4-6 µm Giardiacyst 8-12 µm x 7-10 µm Warsaw March 2009 15

  16. Dr. K. Luden Challenges Warsaw March 2009 16

  17. Dr. K. Luden Performance characteristics • Recovery rates, detection limit, matrix effects • Method does not very well distinguish between viable and nonviable or infectious and non infectious cells • Always analysis of several samples necessary: • spiked sample (positive control, determination of recovery) • natural sample • reliable negative control (exclusion of crosscontamination of the equipment, evaluation of background fluorescence...) Costs ~ 400 €(filter capsule, staining, equipment, hands-on time) Warsaw March 2009 17

  18. Dr. K. Luden German situation Quality controls: proficiency testing? In Germany only very few laboratories applying the method (e.g. Federal environmental agency) No proficiency testing available (how to ship 10 l water samples?) Most of the drinking water from old ground water: very protected water source. No need for analysis but also no possibility for quick implementation of the method at laboratory level. Warsaw March 2009 18

  19. Dr. K. Luden Cryptosporidiosis in Lower Saxony Number of cases reported Warsaw March 2009 19

  20. Dr. K. Luden Cryptosporidiosis in Lower Saxony Lower Saxony 7.9 Million inhabitants 1 incident reported per 50.000 inhabitants and year 90 % of drinking water from protected ground water sources (“old” ground water) 10 % of drinking water influenced by surface waters Warsaw March 2009 20

  21. Dr. K. Luden Questions for health authorities • In case of Cryptosporidiosis • water supply (public, private well, ground water, treated water...) • Living area (rural, farm…) / contact to cattle manure.. • contact to a person suffering from diarrhea • visit at farm with livestock • contact to calfes, pets, non pasteurized milk • playground • public swimming pool Warsaw March 2009 21

  22. Dr. K. Luden Potential contamination of faecal origin • Occurrence of parasites possible but no laboratory capacity for analysis available • Important information on water supply as a basis for risk assessment. • Sampling technique/Sampling point • Distribution system (pipes etc.) • Type of water distributed / water source • Treatment (waterworks, every single step) • Water catchment area Water safety plan / Multi barrier system Warsaw March 2009 22

  23. Dr. K. Luden Thank you for your attention! Warsaw March 2009 23

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