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Summary
Assessing the risk of infectious Cryptosporidium
in drinking water
Pa ul A. R o che lle , Anne M . J o hns o n, Ri car do De L e o n , a n d G e o rg e D. D i G io v a n n i
http://dx.doi.org/10.5942/jawwa.2012.104.0063
Treated drinking water from 14 surface water treatment
plants serving a combined population of approximately 9
million people was monitored for infectious Crypto­
sporidium oocysts using immunofluorescence microscopy
to detect infections in cell culture. No infectious oocysts
were detected in 349,053 L from 370 samples. The
calculated risk of waterborne Cryptosporidium infection
based on the lack of positives, total volume sampled, and
assumptions about daily volume of water consumption
was < 1 in 10,000. However, further study to establish
more accurate risk assessments of the threat to public
health posed by Cryptosporidium in drinking water is still
needed, because results from 14 plants cannot be
extrapolated to the entire United States. This study
demonstrated that routine infectivity monitoring of large
volumes of treated drinking water is feasible. Consequently,
testing finished water for infectious Cryptosporidium
should be considered during periods of regulatory
monitoring, such as the second round of the Long Term 2
Enhanced Surface Water Treatment Rule (LT2ESWTR).
Waterborne cryptosporidiosis is a serious public health
concern, but after two decades of research and monitoring, the water industry still does not have an accurate
assessment of the risk to public health from Cryptospo­
ridium oocysts in drinking water. Although correctly
operating filtration plants usually remove oocysts, the
risk of infection depends on several factors, and there is
uncertainty about the true public health burden from
Cryptosporidium oocysts in drinking water.
During monitoring for the Information Collection
Rule, 1% of treated water samples contained Cryptospo­
ridium oocysts. However, accurately assessing the risk of
waterborne cryptosporidiosis requires quantifying the
prevalence of infectious oocysts in drinking water. In
vitro cell culture combined with polymerase chain reaction detected infectious Cryptosporidium oocysts in 1.4%
of finished water samples from 82 conventional US treatment plants (Aboytes et al, 2004). The results translated
to an annual cryptosporidiosis risk of 52 infections per
10,000 people, which is much higher than the 1 in 10,000
risk of infection goal set by the US Environmental Protection Agency. However, more data on the prevalence of
infectious Cryptosporidium oocysts in drinking water are
needed. The objective of this study was to assess the
prevalence of infectious oocysts in large-volume samples
of treated water using a cell culture assay.
Three of these plants used lakes as source waters; the
remainder used rivers. Filters were shipped to the two cell
culture–processing laboratories by overnight courier in
coolers containing ice packs. Samples were concentrated
using method 1623 (USEPA, 2005) modified for large
volumes of finished water and to allow for infectivity
analysis by cell culture. The final magnetic bead–oocyst
complex was dissociated in an acidified salt solution containing 1% trypsin. Concentrates were inoculated onto
HCT-81 human cell culture monolayers and incubated at
37°C for 64–72 h in a 5% carbon dioxide–humidified
incubator. Infections were detected by applying an antiCryptosporidium sporozoite antibody and observed by
epifluorescence microscopy (Figure 1; Johnson et al, 2012).
METHODS
Treated drinking water samples from 14 plants throughout the United States were filtered onsite by utility personnel.
A full report of this project, Detection of Infectious Cryptosporidium in Conventionally Treated Drinking Water (3021), is
available for free to Water Research Foundation subscribers by
logging on to www.waterrf.org.
RESULTS AND DISCUSSION
Two laboratories analyzed 370 samples of treated
water over a two-year period. Sample volumes ranged
from 84 to 2,282 L, with an average of 943 L for a total
volume of 349,053 L. None of the treated water samples
produced infections. Positive control infections, matrix
spike recovery studies with samples from all participating
utilities, blind matrix spikes, and infections with naturally
occurring oocysts in wastewater all demonstrated that
the oocyst recovery and cell culture procedures performed
as expected and should have detected infectious oocysts
if present. The mean recovery efficiency for matrix spikes
using freshly shed infectious oocysts and processed
through the entire procedure, including cell culture, was
80% (n = 51). The absence of infectious oocysts in these
samples may reflect improved source water protection
and optimization of treatment processes.
Recruiting utilities for the study proved difficult
because of concerns over the potential consequences of a
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2012 © American Water Works Association
79
positive result. The treated water survey was conducted
during the period covered by Cryptosporidium monitoring under the LT2ESWTR for Schedule 1 utilities. Consequently, there was heightened sensitivity to Cryptospo­
ridium-related issues among utility personnel, highlighting
the difficulty of conducting research studies when there
are potential regulatory, public health, legal, and public
relations consequences associated with a positive result.
It is likely that the participating utilities did not anticipate
many positive samples. A different group of utilities with
a broader diversity of water quality characteristics,
greater vulnerability of their source waters to contamination, and less rigorous treatment procedures and controls
might have produced some positive samples.
The 24 positive samples detected in the Aboytes et al
(2004) study translated to an annual cryptosporidiosis risk
of 52 infections per 10,000 people. In the current study, zero
detection of infectious oocysts in 349,053 L equated to an
annual risk of < 1 in 10,000, suggesting that the risk of
Cryptosporidium infection from drinking water produced
by correctly operating treatment plants may be lower than
previously estimated. Much larger volumes were analyzed in
the current study, but the earlier study examined many more
samples, which may have increased the likelihood of capturing infectious oocysts. Therefore, the number of plants,
number of samples, and total volume analyzed must be
considered in any followup infectious Cryptosporidium
monitoring programs to ensure statistically relevant results.
The diversity of Cryptosporidium species and genotypes that can infect HCT-8 cells and be detected using
immunofluorescence is unknown, although the cell culture–immunofluorescence procedure used in the current
study detected infection of HCT-8 cells by C. parvum, C.
hominis, and C. meleagridis oocysts. Therefore, from a
public health perspective, the method detects the most
important human-infectious species.
infection (Aboytes et al, 2004). In the current study, using
a similar oocyst recovery method but larger volumes of
water from fewer treatment plants and a different infection-detection assay, no infectious oocysts were detected.
This absence of infectious oocysts resulted in a calculated
cryptosporidiosis risk of < 1 in 10,000. The results of this
study highlight the difficulty of detecting rare events in
treated drinking water that usually meets infection-risk
goals. However, the results from 14 plants—treating source
waters with low oocyst concentrations—cannot be extrapolated to the entire country. Clearly, better estimates are
needed to accurately assess the threat to public health
posed by Cryptosporidium in drinking water.
Incorporating a cell culture–based infectivity method
into routine and widespread monitoring programs for all
utilities is not practical because of the low prevalence of
infectious oocysts resulting in a high proportion of negative
samples, and the labor and cost of analyzing samples.
However, during the past decade, in vitro cell culture has
gained wider acceptance as a practical method for assessing
infectivity of Cryptosporidium. This study demonstrated
that routine infectivity monitoring of large volumes of
treated water is possible; therefore, the method could be
used to analyze finished water from selected treatment
plants during periods of regulatory monitoring such as the
second round of monitoring under the LT2ESWTR. Monitoring is required to determine whether infectious oocysts
are always present at low levels in all treated water, sometimes present in all treatment plant effluents, or only sometimes present in a fraction of plant effluents.
Acknowledgment
The authors gratefully acknowledge funding from the
Water Research Foundation and thank all participating
utilities that provided filtered samples, water quality data,
and source water oocyst data.
CONCLUSIONS
FOOTNOTE
A previous study on the prevalence of infectious Crypto­
sporidium in treated water concluded that conventional
water treatment does not adequately control the risk of
References
FIGURE 1
Infectious foci (intracellular life stages) of Cryptosporidium hominis (part A) and Cryptosporidium
parvum (part B) in HCT-8 cell cultures detected
by immunofluorescence microscopy
A
B
1ATCC
CCL-244 cell line, American Type Culture Collection, Manassas, Va.
Aboytes, A.; Di Giovanni, G.D.; Abrams, F.A.; Rheinecker, C.; McElroy, W.;
Shaw, N.; & LeChevallier, M.W., 2004. Detection of Infectious Cryptosporidium in Filtered Drinking Water. Jour. AWWA, 90:9:88.
Johnson, A.M.; Di Giovanni G.D.; & Rochelle, P.A., 2012. Comparing
Assays for Sensitive and Reproducible Detection of Cell Cultureinfectious Cryptosporidium parvum and Cryptosporidium hominis in
Drinking Water. Appl. & Envir. Microbiol., 78:156.http://dx.doi.
org/10.1128/AEM.06444-11.
USEPA (US Environmental Protection Agency), 2005. Method 1623 for
Detection of Cryptosporidium and Giardia in Water. EPA 815-R-05002, Ofce. of Water, Washington.
Corresponding author: Paul A. Rochelle is the Source
Water Microbiology Team manager, Metropolitan
Water District of Southern California, 700 Moreno
Ave., La Verne, CA 91750; [email protected].
Scale bar = 100 µm.
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MAY 2012 | JOURNAL AWWA • 104:5 | ROCHELLE ET AL
2012 © American Water Works Association