Warsaw March 2009 1

Dr. K. Luden

Cryptosporidia and Giardia

analytics and challenges

Dr. Katrin Luden

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 2009 2

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

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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).

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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

Dr. K. Luden

Limit values and methods

Cryptosporidium Giardia

Mentioned in directive and ordinance

Not mentioned in directive or ordinance

No parametric value No parametric values

No method No method

Risk assessment ???

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Methods regularly applied in US and UK: US EPA 16223 and UK (DWI), similar to ISO 15553

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

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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

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Concentration

Dr. K. Luden

Warsaw March 2009 9

Sampling site (source water, treated water, tap)

Filter capsuleEnvirocheck, Filta Max

Flow meter(oocysts/volume determination)

Discharge

Pump

peristaltic regular

Concentration

Dr. K. Luden

Filter capsule is washed and elution buffer collected in centrifugation tubes

Centrifugation

Supernatant discarded

Pellet -purification/IMS

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Concentration

Dr. K. Luden

Filter capsule is washed and elution buffer collected in centrifugation tubes

Centrifugation

Supernatant discarded

Pellet -purification/IMS

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Purification by IMS

Dr. K. Luden

Immunomagnetic separation

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YParamagnetic bead

Anti-cryptosporidium-antibodies

Oocyst

Y

Complex separated from debris using magnets and washing

IMS

Dr. K. Luden

Warsaw March 2009 13

Purification by IMS/Detection

Dr. K. Luden

Immunomagnetic separation

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Dissociation of the complex by acid-(heat) treatmentY Y

Staining with FITC labelled antibodies

Detection by microscopy

Epifluorescence microscopy and DIC (Differential interference contrast)

Dr. K. Luden

Warsaw March 2009 15

Cryptosporidium oocyst

4-6 µm

Giardiacyst

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

Dr. K. Luden

Challenges

Step in analysis challenge

Filtration~ 100 % retention but

only 10-80 % recovery

ElutionLoss of oocysts/cysts by incomplete transfer

CentrifugationLoss of oocysts/cysts by disturbing the pellet

Purification by IMSLoss at wash steps

Antibodies specific only to some species

Detection by epifluorescenceBackground fluorescence of debris

Not easily identified because of the common shape

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Dr. K. Luden

Performance characteristics

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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)

Dr. K. Luden

German situation

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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.

Dr. K. Luden

Cryptosporidiosis in Lower Saxony

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Nu

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ase

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5

10

15

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25

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35

Jan Feb Mar April May June July Aug Sept Oct Nov Dec

2005

2006

2007

2008

Dr. K. Luden

Cryptosporidiosis in Lower Saxony

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Year cases reported2005 1532006 1452007 1442008 161

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

Dr. K. Luden

Questions for health authorities

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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

Dr. K. Luden

Potential contamination of faecal origin

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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

Dr. K. Luden

Thank you for your attention!

Warsaw March 2009 23