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
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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
mb
er
of c
ase
s re
po
rted
0
5
10
15
20
25
30
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!
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