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Cryptosporidium and Giardia: Assessment of the Efficiency of a Drinking Water Treatment Plant

Thursday, 29 September, 2005 - 17:00
Campus: Brussels Humanities, Sciences & Engineering campus
Faculty: Science and Bio-engineering Sciences
Stéphane Mazoua
phd defence

The importance of waterborne transmission for Cryptosporidium and
Giardia was recognised some 15 years ago, and since then an important
number of waterborne outbreaks of cryptosporidiosis and giardiasis have
been documented throughout the world (Centers for Disease Control,
1990, 1994, 1997, 2000; Joce et al., 1991; LeChevallier et al., 1991;
LeChevallier et al., 1991; Mac Kenzie et al., 1994; Van Asperen et al.,
1996; Kramer et al., 1998; Guyonnet et al., 2002). Cryptosporidium
oocysts and Giardia cysts occur commonly in the aquatic environment,
and great interest is shown by the water industry, worldwide, in detecting
waterborne cysts and oocysts and limiting the transmission of these
waterborne protozoan parasites.

As the primary goal of drinking water suppliers is to provide water free of
microbial and chemical contaminants, the emergence of parasitic protozoa
such Cryptosporidium and Giardia as etiological agents of waterborne
disease has prompted renewed evaluation of the efficiency of water
treatment processes. Increasingly stringent regulations for drinking water
quality, in particular in the United States and in the United Kingdom, also
require effective removal of these organisms.

A first challenge is posed by the development of a method to accurately
detect and enumerate these pathogenic micro-organisms. The method we
developed in CIBE's laboratory was largely based on USEPA Method 1623
and consists of filtration of the water sample through a filtration cartridge
followed by immuno-magnetic separation and immunofluorescence.
Although several steps of the method generated significant losses, this
method proved efficient for detecting Cryptosporidium and Giardia in
drinking water, provided that the right filtration cartridge is chosen and all
steps are optimised in laboratory. The method gave recoveries of 85 ±
19% for Cryptosporidium and 57 ± 11% for Giardia on tap water using
Envirochek(r) HV cartridges, whereas Filta-Max(tm) cartridges provided
recoveries of 44 ± 15% and 63 ± 9% for Cryptosporidium and Giardia

Our experiments showed that Envirochek(r) HV cartridges were the most
efficient to analyse simultaneously Cryptosporidium and Giardia, provided
that the elution of the cartridge is performed as recommended by the
supplier, with addition of a vertical agitation 3 times for 10 minutes
followed by a vortex agitation (30 seconds) for each extremity of the
capsule. Then, the cartridge should be strongly shaken up-down manually
in order to extract as much eluent as possible. For the analysis of Giardia,
Filta-Max(tm) and Envirochek(r) HV cartridges provided equivalent results
with this procedure. However, better results could be obtained for Giardia
during the elution step if the Envirochek(r) HV cartridge was eluted as
recommended by the supplier. This suggests that the optimisation of the
detection method could be more efficient if developed for the single
detection of each micro-organism. Envirochek(r) cartridges were also
tested for both micro-organisms, but provided lower recoveries.

As regards the concentration of the cartridges eluent, centrifugation and
filtration gave equivalent results provided that centrifugation parameters
were increased until 2 700xg for 30 minutes instead of 1 100xg for 15
minutes as recommended by USEPA Method 1623. However, as filtration
seemed more likely to be further improved by the use of other types of
membranes, it was retained for the following experiments. Immunomagnetic
separation was optimised by increasing the volume of acid to
dissociate the micro-organism/bead complex (100 μL instead of 50 μL
recommended by the supplier of magnetic beads). This led to improved
recoveries for both micro-organisms. In contrast, increasing the volume of
magnetic beads or changing the pH of the PBS solution used for immunomagnetic
separation did not have a significant impact on recoveries,
neither did an increase of the agitation time. Immuno-fluorescent staining
was improved by using methanol instead of acetone as fixative reagent,
which led to better results for Giardia. Moreover, our investigations
showed that the drying time before fixation is a critical factor and should
be monitored carefully.

With all steps optimised, the detection method generated losses of around
15% of Cryptosporidium oocysts with the Envirochek(r) HV cartridge and
around 40% of Giardia cysts with both Envirochek(r) HV and Filta-
Max(tm) cartridges. Further improvement of Giardia recoveries was
obtained at a later stage by increasing the volume of acid during immunomagnetic
separation. It should also be noted that the skills of the
experimentalist improved significantly after having processed numerous
samples, resulting in an improvement of the recovery results. This
confirms the fact that analyst skills are a critical factor affecting the
efficiency of the method, as observed commonly.

Overall, the method proved tedious and time-consuming, in particular as
regards the microscopic examination of the slides. Significant
improvement could be obtained through the development of an automatic
enumeration procedure. Encouraging results have been published recently
in this field (Widmer et al., 2005). The recent production of
Cryptosporidium reference materials should also contribute to further
improvement of each step of the method.

Our study revealed the difficulties to reach good recoveries for quantifying
Cryptosporidium and Giardia on environmental water samples. When
applied to raw water from the river Meuse and to water taken at the outlet
of the filtration stage of the drinking water treatment plant, the recovery
of the method conducted with Filta-Max(tm) cartridges decreased
significantly (43 ± 15% for Cryptosporidium and 54 ± 33% for Giardia on
river Meuse water, and 10 ± 1 and 12 ± 7% respectively on filtered
water). Therefore, the efficiency needs to be determined for each type of
water matrix.

For more information: see the attachments.