1. Warsaw March 20091 Dr. K. Luden Cryptosporidia and Giardia analytics and challenges Dr. Katrin Luden

2. Dr. K. Luden Cryptosporidium  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 Warsaw March 20092

3. Dr. K. Luden Giardia  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 Warsaw March 20093

4. Drinking water directive 98/83/EC 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). Warsaw March 20094

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 20095

6. Dr. K. Luden Limit values and methods CryptosporidiumGiardia Mentioned in directive and ordinance Not mentioned in directive or ordinance No parametric valueNo parametric values No method Risk assessment??? Warsaw March 20096 Methods regularly applied in US and UK: US EPA 16223 and UK (DWI), similar to ISO 15553

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 20097

8. Dr. K. Luden Sampling: 10 to 1.000 l of water – into transport containers (grab samples) or filtering in the field flow 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 20098

9. Concentration Dr. K. Luden Warsaw March 20099 Sampling site (source water, treated water, tap) Filter capsule Envirocheck, Filta Max Flow meter (oocysts/volume determination) Discharge Pump peristaltic regular

10. Concentration Dr. K. Luden Filter capsule is washed and elution buffer collected in centrifugation tubes Centrifugation Supernatant discarded Pellet -purification/IMS Warsaw March 200910

11. Concentration Dr. K. Luden Filter capsule is washed and elution buffer collected in centrifugation tubes Centrifugation Supernatant discarded Pellet -purification/IMS Warsaw March 200911

12. Purification by IMS Dr. K. Luden Immunomagnetic separation Warsaw March 200912 Y Paramagnetic bead Anti-cryptosporidium- antibodies Oocyst Y Complex separated from debris using magnets and washing

13. IMS Dr. K. Luden Warsaw March 200913

14. Purification by IMS/Detection Dr. K. Luden Immunomagnetic separation Warsaw March 200914 Dissociation of the complex by acid- (heat) treatment Y Y Staining with FITC labelled antibodies Detection by microscopy

15. Epifluorescence microscopy and DIC (Differential interference contrast) Dr. K. Luden Warsaw March 200915 Cryptosporidium oocyst 4-6 µm Giardia cyst 8-12 µm x 7-10 µm Detection Counterstaining with DAPI is recommended to detect nuclei of sporozites and trophozoites DIC detects internal morphological characteristics

16. Dr. K. Luden Challenges Step in analysischallenge Filtration ~ 100 % retention but only 10-80 % recovery Elution Loss of oocysts/cysts by incomplete transfer Centrifugation Loss of oocysts/cysts by disturbing the pellet Purification by IMS Loss at wash steps Antibodies specific only to some species Detection by epifluorescence Background fluorescence of debris Not easily identified because of the common shape Warsaw March 200916

17. Dr. K. Luden Performance characteristics Warsaw March 200917 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)

18. Dr. K. Luden German situation Warsaw March 200918 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.

19. Dr. K. Luden Cryptosporidiosis in Lower Saxony Warsaw March 200919 Number of cases reported

20. Dr. K. Luden Cryptosporidiosis in Lower Saxony Warsaw March 200920 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

21. Dr. K. Luden Questions for health authorities Warsaw March 200921 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

22. Dr. K. Luden Potential contamination of faecal origin Warsaw March 200922 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

23. Dr. K. Luden Thank you for your attention! Warsaw March 200923