United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Research Triangle Park NC 27711
Research and Development
EPA-600/S1-83-014 Oct 1983
&EPA Project Summary
Determination of the Use of Solid
Particle Samplers for Giardia
Cysts in Natural Waters
William S. Brewer
Parasitic flagellates in the genus
Giardia are distributed worldwide and
are now the most commonly reported
intestinal parasites in the United States
and Britain. Twenty-three waterborne
outbreaks of giardiasis affecting over
7000 people occurred in various states
in the United States between 1965 and
1977. Because of this significant
increase in the incidence of waterborne
outbreaks of giardiasis, efforts have
been made to develop reliable and/or
sensitive methods to determine the
presence or absence of G. lamblia cysts
in water supplies. The primary objective
of this study was to improve the current
methodology for concentrating, re-
covering and detecting cysts of G.
lamblia in water supplies.
Two sampling processes for the
concentration of cysts were examined.
One process was diatomaceous earth
filtration while the second was that of
cyst concentration onto charged par-
ticles. Cysts of G. muris were used to
determine the retention efficiency of ion-
exchange resins and each type of
diatomaceous earth filter examined.
Cyst desorption efficiencies were
evaluated for ion-exchange resins that
best retained cysts, while backwashing
parameters were optimized for diato-
maceous earth filters. Results of cyst
retention experiments indicated that
two processes, anion-exchange con-
centration of cysts and diatomaceous
earth filtration, had the potential to be
developed into field methods. Compari-
son of these two processes at low cyst
inoculum concentrations (1 x 103
cysts/liter) indicated that a greater
number of cysts could be recovered
from the diatomaceous earth filters.
When 40 liter samples of tap water con-
taining between 1.0 x 104 and 7.0 x 10s
cysts were passed through diato-
maceous earth filters. 5.2 to 31.1 % of the
cysts were recovered in the backwash.
As a result, the diatomaceous earth
filter was comparable to microporous
filtration and may have application in
sampling finished water supplies.
However, its utility in raw sampling was
limited since turbidity severely reduced
the recovery efficiency of cysts.
This Project Summary was developed
by EPA's Health Effects Research
Laboratory. Research Triangle Park,
NC. to announce key findings of the
research project that is fully documented
in a separate report of the same title (see
Project Report ordering information at
back).
Introduction
The primary objective of this study was
to improve the current methodology for
concentrating, recovering, and detecting
cysts of G. lamblia in water supplies. Two
sampling processes for concentration of
G. lamblia cysts were examined. One
process was diatomaceous earth filtration
while the second process examined was
that of cyst concentration onto charged
particles. The first process was based on
the hypothesis that cysts could be
efficiently trapped on the surface of
diatomaceous earth filters and sub-
sequently recovered through backwashing
the filter with a small volume of water.
The second process was based on the
hypothesis that cysts could be attracted to
charged surfaces, since they have been
shown to have a charge of approximately
-25 mV at pH 5 5 and to increase their
electronegativity as the pH rises to 8 0 In
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addition, charge-attraction techniques
have been applied to concentration of
viruses and bacteria from water, to the
concentration of trypanosomes, and to
the concentration of Plasmodium in
clinical samples.
Parasitic flagellates in the genus
Giardia are distributed worldwide and are
now the most commonly reported human
intestinal parasites in the United States
and Britain. The cycle of this parasite is
composed of two stages: the cyst stage
and the trophozoite stage. Transmission
of Giardia most often occurs when viable
cysts are ingested directly or through
water contaminated with feces. The
average incubation period for human
giardiasis is 8 days, with a range of 3-42
days. While most infections are asymp-
tomatic, some people have a snort-
lasting acute diarrheal disease, nausea,
and anorexia. A small percentage develop
an intermittent or protracted course
characterized by diarrhea, cramping,
abdominal pain, bloating, and flatulence.
Diarrhea with or without overt malab-
sorption may last months or even years.
As a measure of the significance of
giardiasis in the United States, 23
waterborne outbreaks affecting over
7000 people have been caused by this
infection between 1965 and 1977.
Epidemic outbreaks have been reported
in New York, New Hampshire, Pennsyl-
vania, Colorado and Washington. The
majority of these outbreaks were attribu-
table to public consumption of minimally
treated water which was fecally con-
taminated. In addition, sporadic epidemics
have occurred among infants and children
in hospital nurseries, custodial and
residential institutions, and in day-care
centers where personal hygiene standards
were not stringent. Water supplies were
most likely contaminated by untreated
human waste or by aquatic mammal
waste since asymptomatic carriers and
aquatic mammals have been identified as
the major reservoirs.
Because of the significant increase in
the incidence of waterborne outbreaks of
giardiasis reported, efforts have been
made to develop reliable and/or sensitive
methods to determine the presence or
absence of G. lamblia cysts in water
supplies. The major problem associated
with developing sampling technology is
that cysts are assumed to be present in
low numbers, therefore necessitating the
need for large-volume water sampling.
During the past few years, several
approaches have been taken to concen-
trate and detect cysts in water supplies.
Developed methods can be divided into
three major categories: (a) membrane
filtration, (b) particulate filtration, and (c)
microporous filtration.
Moore et al. (1969) utilized membrane-
filtration to examine water and sewage
samples for Giardia cysts. One- and two-
liter samples were passed through
cheesecloth and then filtered through
0.45 /urn porosity membrane filters.
Sediment on the filter surface was
brushed into water, centrifuged and
preserved in 10% formalin for microscopic
examination. No cysts were observed in
10 water samples. Barbour and his
coworkers (1976) used the method of
Chang and Kabler to filter 22 liters of
stream water following an outbreak.
However, no Giardia cysts were found.
Luchtel and colleagues (1980) utilized
293 mm diameter 5.0 /urn pore size
Nuclepore* filters to concentrate formalin-
fixed G. lamblia cysts from 20 liter tap
water samples. Recovery rates of approx-
imately 75% were found under such
conditions.
The Center for Disease Control de-
veloped a large-volume sampling tech-
nique. The method used a swimming
pool filter in which sand was the
sampling medium. During an outbreak in
Rome, New York, a total volume of 1.1 x
106 liters (28,000 gal.) of water was
collected at an average flow rate of 76
liters/minute (20 gal./min) through the
filter daily for 10 days. Thef ilter backwash
was collected each day in two 210-liter
drums and coagulated with alum. The
resulting sediment was collected and
aliquots were fed to beagle puppies and
examined microscopically. Two of ten
samples fed to dogs produced infection
and a single cyst was observed in one
sample. In 1976, the U.S. Environmental
Protection Agency (EPA) developed a cyst
concentration technique involving the
filtration of a large volume of water (100
gal. or more) through a microporousorlon
fiber filter. This method has been
tentatively adopted as the "method of
choice" for concentrating cysts from
water supplies. However, the reliability
and validity of the technique has yet to be
fully evaluated. Limited laboratory evalu-
ations have indicated that cyst recovery
was only in the range of 3-15%, with a
mean of 6.3%. The interpretation of
positive or negative field data is uncertain.
In the sampling methodology described
above, efficient cyst concentration from
water was possible under certain condi-
tions. However, processing of the con-
centrate led to significant losses of cysts.
Therefore, a major problem in detecting
Giardia cysts in contaminated water
supplies using any of the above techniques
has been the quantitative recovery of
cysts from the filter medium.
Summary
The feasibility of using diatomaceous
earth filters or ion-exchange resins to
concentrate cysts of G. lamblia from
water samples and subsequently recover
those cysts in a quantitative manner was
evaluated with water samples experi-
mentally contaminated with G. muris
cysts. A series of ion-exchange resins
were initially selected for evaluation.
Anionic resins selected included DEAE-
cellulose and two polystyrene, divinyl-
benzene-crosslinked resins, Dowex 1-
X4* and Dowex 1-X8. Cationic resins
evaluated included Dowex 50W-X4 and
Dowex 50W-X8. The relative ability of
each resin to capture cysts from water
samples was determined by passing cysts
in buffered, distilled water through
ionically-charged columns and determin-
ing the fraction of cysts retained at
varying inoculum concentrations. DEAE
cellulose retained all the cysts passed
through the column regardless of the
inoculum concentration; whereas, Dowex
1-X4, Dowex 1-X8, Dowex 50W-X4, and
Dowex 50W-X8 retained 101,71,75, and
97% of the inoculated cysts, respectively.
Statistical analysis of the data indicated
that there was no significant difference
between the ability of DEAE cellulose,
Dowex 1-X4 and Dowex 50W-X8 to
capture cysts from water samples.
Therefore, these resins were selected for
desorption studies. Cysts concentrated
on the surfaces of ion-exchange resins
were eluted with 40 ml of buffer optimized
to a particular pH and ionic strength (plj
and the percent recovered from each
resin was compared. Approximately 49°A
of the cysts concentrated on Dowex 1-X4
resins could be recovered throughout th<
inoculum range tested; however, ar
average of only 38% of the inoculatec
cysts were recovered from the Dowe)
50W-X8 columns. No cysts were recoveree
from DEAE cellulose resins regardless o
the elution pH or pi.
The capacity of three types of diato
maceous earth filter, Celite 505, Hyflo
SuperCel, and Celite 560, to concentrati
cysts was evaluated by passing distillei
water samples containing between 6.(
cysts/liter and 1.62 x 104 cysts/lite
through each column. No significan
differences between the diatomaceou
earth filters were observed, with retentioi
ranging between 66 and 100%. Howevei
'Mention of trade names or commercial produc
does not constitute endorsement or recommendatic
for use
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significant differences between columns
were noted when each was backwashed
to recover the cysts captured on the
surface. Best cyst recoveries were
observed when the filters were back-
washed with 2000 ml of distilled water at
a flow rate of 2 liters/minute. An average
of 13% of the cysts concentrated on Celite
560 columns could be recovered. How-
ever, because of the concentration of
small diatomaceous earth particles or
"fines" in the backwash of both Celite
505 and Hyflo-SuperCel columns, no
cysts could be detected under the micro-
scope. Therefore, Celite 560 was selected
for comparative studies with Dowex 1-
X4.
The utility of either Dowex 1-X4 or
diatomaceous earth (Celite 560) columns
as Giardia cyst sampling devices was
compared by sampling 40 liter distilled
water samples contaminated with cysts
and comparing the number of cysts
recovered from each sampler. The mean
number of cysts recovered was not
significantly different over the range of
inoculum concentrations used; however,
analysis of the data indicated that there
was a higher probability of recovering cysts
from water samples containing low
concentrations of cysts (1.0-1.5 x 103
cysts/liter) when the diatomaceous earth
filter was used. Cyst recoveries from
water samples passed through diato-
maceous earth averaged 13%, similar to
that observed when microporous filtra-
tion was evaluated. However, the effi-
ciency of the diatomaceous earth filter
was markedly decreased when turbid wa-
ter was sampled. When the recovery of
cysts inoculated into turbid water sam-
ples (24 FTU) was compared to that of tur-
bid-free samples, an 85-86% reduction
was observed.
Conclusions
Based on the objectives and results of
this study the following conclusions can
be drawn: 1) Results of resin capacity and
cyst retention experiments indicated that
two processes, anion-exchange concen-
tration of cysts and diatomaceous earth
filtration, had the potential to be devel-
oped into field methods. 2) Analysis of the
data with respect to inoculum concentra-
tion indicated that a greater number of
cysts were recovered from the diatoma-
ceous earth filters when dilute samples (1
x 103 cysts/liter) were filtered. Based on
these data diatomaceous earth filters
were considered the best choice. 3) Com-
parison of the efficiency of the diatoma-
ceous earth filter to the reported efficien-
cy of the EPA method of microporous fil-
tration (13.0 and 6.7%, respectively) indi-
cated that the two were similar. However,
it appeared that the diatomacous earth
filter was more severely affected by the
composition of the water sample than the
microporous filter would be. 4) The diato-
maceous earth filter may have applica-
tion to finished water supplies; however,
its use on raw water samples is limited at
the present time.
Recommendations
1. The results of this study indicate that
Giardia cysts could be efficienctly con-
centrated on either the surface of
diatomaceous earth or on the surfaces of
charged particles. However, subsequent
research should be directed toward the
efficient recovery of concentrated cysts.
Further research should be carried out
with weak ion-exchange resins that
operate efficiently in a narrower pH range
or with charge-modified filters similar to
those used to concentrate viruses.
2. One of the major problems encoun-
tered in this study was the lack of a
sensitive detection technique. Quantifi-
cation of Giardia cysts is presently based
upon microscopic identification and
counting. Experimental studies should be
carried out to develop accurate detection
of small numbers of cysts mixed with
other microorganisms and debris from
aquatic habitats. Immunofluorescent
techniques similar to those used for the
detection of bacteria should be investi-
gated.
3. The behavior of Giardia cysts on
certain resins observed in this study
raised some question concerning the
biochemical and physiological nature of
cysts. Little information is available on
these subjects. Basic research on the
nature of the cysts themselves would
lead to rational decisions on applicable
sampling methods.
References
Barbour, A.G., C.R. Nichols, andT. Fuku-
shina. 1976. An outbreak of giardiasis in
a group of campers. Am. J. Trop. Med.
Hyg. 25; 384-389.
Luchtel, D.L., W.P. Lawrence, and F. B.
DeWalle. 1980. Electron microscopy of
Giardia Iambi/a cysts. Appl. Environ.
Microbiol. 40: 821-832.
Moore, G.T., W.M. Cross, D. McGuire,
C.S. Mallohan, N.N. Gleason, G.R. Healy,
and LH. Newton. 1969. Epidemic giard-
iasis of a ski resort. N. Eng. J. Med. 281:
402-407.
William S. Brewer is with Wright State University, Dayton. OH 45434.
Frank W. Schaefer, III is the EPA Project Officer (see below).
The complete report, entitled "Determination of the Use of Solid Particle Samplers
for Giardia Cysts in Natural Waters," (Order No. PB 83-246 090; Cost: $10.00,
subject to change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park. NC 27711
•ftUS GOVERNMENT PRINTING OFFICE 1983-659-017/7207
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