APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 2011, p. 5420–5427 Vol. 77, No. 150099-2240/11/$12.00 doi:10.1128/AEM.00416-11Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Rapid Detection and Enumeration of Giardia lamblia Cysts in WaterSamples by Immunomagnetic Separation and

Flow Cytometric Analysis�†Hans-Anton Keserue,1,2,3 Hans Peter Fuchslin,1 and Thomas Egli1,3*

Eawag, Swiss Federal Institute for Aquatic Science and Technology, Uberlandstr. 133, P.O. Box 611, CH-8600 Dubendorf, Switzerland1;Federal Office of Public Health (FOPH), Schwarzenburgstrasse 165, CH-3097 Liebefeld, Switzerland2; and Institute of

Biogeochemistry and Pollutant Dynamics (IBP), ETH Zurich, Universitatsstrasse 16, 8092 Zurich, Switzerland3

Received 24 February 2011/Accepted 5 June 2011

Giardia lamblia is an important waterborne pathogen and is among the most common intestinal parasites ofhumans worldwide. Its fecal-oral transmission leads to the presence of cysts of this pathogen in the environ-ment, and so far, quantitative rapid screening methods are not available for various matrices, such as surfacewaters, wastewater, or food. Thus, it is necessary to establish methods that enable reliable rapid detection ofa single cyst in 10 to 100 liters of drinking water. Conventional detection relies on cyst concentration, isolation, andconfirmation by immunofluorescence microscopy (IFM), resulting in low recoveries and high detection limits. Manydifferent immunomagnetic separation (IMS) procedures have been developed for separation and cyst purification,so far with variable but high losses of cysts. A method was developed that requires less than 100 min and consistsof filtration, resuspension, IMS, and flow cytometric (FCM) detection. MACS MicroBeads were used for IMS, anda reliable flow cytometric detection approach was established employing 3 different parameters for discriminationfrom background signals, i.e., green and red fluorescence (resulting from the distinct pattern emitted by thefluorescein dye) and sideward scatter for size discrimination. With spiked samples, recoveries exceeding 90% wereobtained, and false-positive results were never encountered for negative samples. Additionally, the method wasapplicable to naturally occurring cysts in wastewater and has the potential to be automated.

Giardia spp. are ubiquitous protozoan intestinal parasitesinfecting the small intestine of humans and various animals.Giardia lamblia (syn. G. duodenalis or G. intestinalis) is the onlyGiardia species found in humans (45) and is the most commonintestinal parasite of humans worldwide (27). The vegetativeform of these parasites, the trophozoites, which detach fromthe intestinal villi, are pear-shaped, 9 to 21 �m long, 5 to 15 �mwide, and 2 to 4 �m thick and have two nuclei and eight flagellaorganized in four pairs (15). The noninvasive life cycle includesexcystation and encystation processes, which take place duringpassage through the intestines. After detachment, the parasitesform cysts that are ovoid, 8 to 14 �m long, and 7 to 10 �m wideand are excreted with the feces (23, 29). This encystation pro-cess involves discontinuance of cell division, but DNA replica-tion is still performed (endoreplication), resulting in cysts with4 nuclei (42). Thus, the exyzoite stage is prepared for directand rapid cell division into four trophozoites (3). This could beone reason for the reported low infection doses, which can beas low as 1 to 10 cysts (36). The incubation time averagesaround 1 week, ranging from 3 to 20 days (31), and patients canexcrete up to 107 cysts per gram of stool (7, 35).

Infection occurs via the fecal-oral route, and besides di-rect transmission (41), the main source of infection is con-

sidered to be contaminated water (24, 26). Additionally,transmission from crops harvested from agriculture fieldstreated with sewage sludge has been reported (16). The zoo-notic character of the disease is still under discussion (44),though many waterborne outbreaks have been associated withzoonotic transmission (22).

The prevalence of human Giardia infection in industrializedcountries ranges from 2% to 5%, with a prevalence of 20% to30% in developing countries, probably due to a less developedsanitation infrastructure in the latter (32).

Giardia cysts are ubiquitous in surface waters worldwide,their concentrations are reported to be in the range of 0.01 to100 cysts per liter (50), and they survive for up to 2 months inwater at 8°C (29). They are reported to be strongly resistant todisinfection, including chlorination, and difficult to remove bystandard filtration (50). In contrast to the case for other wa-terborne pathogens, no tolerance values for drinking water areestablished (19), but based on known breakouts of giardiasis,action measures for levels of 3 to 5 cysts/100 liters are proposed(49). Thus, the requirement of detecting 1 cyst per 10 to 100liters of water has been expressed (12), and sensitive and reli-able methods are required.

Excystation and culture protocols have been developed; nev-ertheless, the organism cannot be cultured reliably from watersamples (10, 37). Thus, classical microbiological approaches,such as plating, are not applicable for this organism.

Approved standard methods for Giardia and Cryptospo-ridium detection in water samples are described in the USEPA1623 and AFNOR NF T 90-455 protocols. Both are very sim-ilar, and most existing methods are based on them (18). Gen-erally, they rely on concentration, purification, and detection

* Corresponding author. Mailing address: Eawag, Swiss Federal In-stitute for Aquatic Science and Technology, Uberlandstr. 133, P.O.Box 611, CH-8600 Dubendorf, Switzerland. Phone: 41 44 823 51 58.Fax: 41 44 823 55 47. E-mail: egli@eawag.ch.

† Supplemental material for this article may be found at http://aem.asm.org/.

� Published ahead of print on 17 June 2011.

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steps, and recoveries of �1% to 61% have been reported,depending on the method, seeding level, and sample turbidity(38). Recently, improved USEPA 1623-based methods showedrecoveries from tap water of up to 75.4% (21).

Though improvements have been achieved in all respectscompared to initially proposed methods, there is still a need forprogress, as the methods are considered tedious, expensive,and only semiquantitative. A blind survey conducted in 16different commercial laboratories performing analyses ofspiked wound filter samples resulted in recoveries rangingfrom 0.8 to 22.3%, averaging 9.3%, which illustrates the above-mentioned drawbacks (6).

Many molecular identification methods based on PCR havebeen proposed, with detection limits ranging between 1 and105 cysts (data are given predominantly in cysts per liter butalso in cysts per PCR), but difficulties due to small amounts oftarget DNA, the presence of inhibitor substances, and false-positive results were reported (4, 25, 28, 39).

Recently, different detection methods using flow cytometry(FCM) were proposed, employing different staining methodsand resulting in variable recovery. They were usually accom-panied by false-positive results (14, 20, 34, 47).

In this study, we developed a fast screening detection assayinvolving the filtration of cysts, immunomagnetic separation(IMS), and flow cytometric detection. The results presentedhere describe a rapid and robust method that can be applied todifferent water samples and has the prospect of being applica-ble to various matrices and to the automation potential of flowcytometry.

MATERIALS AND METHODS

Giardia lamblia cysts. G. lamblia cysts were obtained from Waterborne Inc.(New Orleans, LA) and stored (107 cysts) in 8 ml of sterile phosphate-bufferedsaline (PBS) (150 mM NaCl, 15 mM KH2PO4, 20 mM Na2HPO4, 27 mM KCl,pH 7.4; Sigma-Aldrich, St. Louis, MO) with 2.5% formalin (Sigma-Aldrich) at4°C. The cysts were from an H-3 human isolate purified after passage throughMongolian gerbils.

Before dilutions were prepared, cyst samples were sonicated for 6 min in anultrasonic bath (TUC-600; M. Scherrer AG, Wil, Switzerland) at 35 kHz and 600W in order to dissociate the majority of the agglomerated cysts. The optimumsonication time was established by calibrating measurements and by evaluationof the results by FCM and immunofluorescence microscopy (IFM), resulting inaround 90% single cysts and 10% agglomerated cysts. Agglomerates consistedmainly of 2 cysts and, rarely, 3 or more cysts sticking together.

Water samples. One-liter tap water samples were collected from different tapsin the laboratory building of the Swiss Federal Institute for Aquatic Science andTechnology (Eawag). The system is fed by the drinking water supply of the townof Dubendorf, Switzerland.

Pond water was collected from a stagnant small pond of around 5 m indiameter in proximity to the laboratory building. River water samples were takenat the Chriesbach River, Dubendorf, Switzerland.

Wastewater samples were obtained from the inlet pipe of the experimentalwastewater treatment plant of the Eawag, which has access to real wastewaterfrom the community of Dubendorf.

All samples were processed directly or stored at 4°C for a maximum of 1 h.Tap and surface water samples. Water samples were collected in 1-liter screw-

cap glass bottles and inoculated with 1 ml filtered (0.22-�m pore size) PBScontaining a defined number of cysts, as indicated in the different experiments.The inoculated water samples were subsequently shaken vigorously. The watersamples were prefiltered through a 30-�m nylon mesh filter (Millipore AG, Zug,Switzerland) before being vacuum filtered through a 5-�m-pore-size polycarbon-ate track etch filter (PCTE; Sterlitech Corporation, Kent, WA). The negativepressure was kept below 103 Pa for tap water filtration, below 3 � 103 Pa for riverwater filtration, and below 4 � 103 Pa for pond water filtration. The differentvacuum pressures were required due to the different turbidities of the watersamples in order to avoid clogging of the filter (see below for more details).

The filter was carefully removed and inserted into a 50-ml centrifugation tube,followed by addition of 5 ml sterile PBS and 50 �l 1% bovine serum albumin(BSA; Fluka), strong vortexing, and addition of 1.5 �l fluorescein-labeled mono-clonal antibodies (MAbs) (Giardi-a-Glo 20� solution; Waterborne Inc.). Afterincubation for 30 min at ambient temperature in the dark, 50 �l of superpara-magnetic anti-fluorescein isothiocyanate (anti-FITC) MicroBeads (Miltenyi Bio-tec GmbH, Bergisch Gladbach, Germany) was added at 4°C. The assay mix wasthen incubated for 30 min at this temperature.

For 100-liter water samples, two inline filter cartridges were attached sequen-tially with rubber tubes to the tap, with the closer one to the tap comprising the30-�m nylon mesh filter and the downstream one comprising the PCTE mem-brane. The flow rate was set to 2 liters per minute. Spiking was maintained byinjecting the cysts through a syringe and a hollow needle through the rubber tubeinto the stream upstream of the filters. Injection was done after 10 min of a totalfiltration time of 50 min. Subsequently, the PCTE membrane was carefullyremoved and treated as described above.

Wastewater preparation. Wastewater samples were prepared as describedabove for the surface water samples, except for the following modifications. Priorto the 30-�m nylon mesh filtration, the samples were filtered through a 1-cm-thick layer of glass wool (Sigma-Aldrich). The negative pressure was maintainedbelow 6 � 103 Pa, and the sample volume was 100 ml. Due to the very rapidclogging of the filter, it was impossible to process 1 liter of sample.

IMS. Immunomagnetic separation using MACS MS columns (Miltenyi BiotecGmbH) was conducted at a defined speed by a peristaltic pump (Alitea U1-Midi-D pump; Bioengineering AG, Wald, Switzerland) at a flow rate of 2.05ml/min. The sample volume of 5 ml was run through the column after placing thecolumn in the separator magnet. Subsequently, the column was washed twice byrinsing with 3 ml of PBS, followed by two washes with 3 ml of PBST (PBS plus 0.01%Tween 20; Fluka) and finally with 3 ml PBS, conducted at a flow rate of 2.39 ml/min.The washing buffers were added to the column just before the columns ran dryaccording to the manufacturer’s instructions. After removal of the column from themagnet, the purified cells were recovered by flushing 1 ml PBS through the columninto a 15-ml Falcon tube by firmly applying the provided plunger.

Turbidity evaluation. The turbidity of samples was measured with a Hach 2100turbidimeter (Hach Company, Loveland, CO) following the instructions of themanufacturer. Results are given in nephelometric turbidity units (NTU).

Microscopic cyst enumeration. Samples for microscopic cyst enumerationwere stained as described above with 1.5 �l specific FITC-coupled surface anti-body (Waterborne). Stained cysts were filtered onto a 13-mm, 0.2-�m-pore-sizeblack PCTE filter (Isopore; Millipore), using a 3-ml syringe and a 13-mm filterholder cartridge. The filter was dried on a glass slide and a small drop of antifademounting medium (Waterborne Inc.) was applied before a cover glass was placedover the filter. Microscopic enumeration was performed using an Olympus IX51inverted microscope equipped with filters for FITC at a magnification of �400.The provided software (Olympus DP-Soft, version 3.2) was applied to analyze 10pictures taken with a defined image area of 1.34 mm2, and the average forcounted cells was extrapolated to the filter perimeter after subtracting the borderarea covered by the filter cartridge sealing, resulting in an effective filter area of86.59 mm2.

The average number of cysts was calculated according to the equation Ct �(�Ci � r2�)/(n � Ai), where Ct is the total number of cysts in the sample, Ci isthe number of cysts counted within the image area, n is the number of images,r is the effectively exposed filter radius, and Ai is the image area.

FCM detection. Flow cytometric detection was performed with a PartecCyFlow space flow cytometer (Partec GmbH, Muenster, Germany) equippedwith a 200-mW blue solid-state laser emitting light at 488 nm. The volumetriccounting properties of the flow cytometer measured the events in 200 �l ofsample volume and extrapolated the result to 1 ml of sample volume. Opticalfilters were adjusted to measure green fluorescence at 520 nm, red fluorescenceat 630 nm, and the side scatter (SSC) at 488 nm. The trigger was set on greenfluorescence. Events were defined based on SSC, 520-nm and 630-nm fluores-cence. Results were presented by plotting green fluorescence (520 nm) versus redfluorescence (630 nm) as well as versus SSC and by applying defined gatingregions.

The R1 gate of the green (520 nm; y axis) versus red (630 nm; x axis) bivariatedot plot and the RN1 gate of the sideward scatter histogram (488 nm) werecombined into an RN1-R1 multigate, thus counting only cysts that lay inside bothrestriction gates (Fig. 1). The specific instrumental gain settings used for greenfluorescence, red fluorescence, and sideward scatter channels were 259, 345, and191.1, respectively. The speed setting was 3, implying a counting rate of �500events s�1.

Data treatment. Giardia cyst enumeration is expressed as the mean for theexperiments (n � 3) � the standard deviation. The recovery or agreement

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quotas, expressed as percentages, represent the quotients of the means of therespective experiments (n � 3) � the normalized root mean square deviations,taking into account the error propagation included in the procedure. Calcula-tions and charts were established with Microsoft Excel 2003.

RESULTS

Spiking experiments. (i) Agreement between flow cytometricand immunofluorescence microscopic counting. A suspensionof G. lamblia cysts in sterile filtered PBS was stained withFITC-coupled surface antibodies and enumerated by micros-copy and flow cytometry (Table 1). The labeled cysts could beidentified clearly by the fluorescence microscope (see Fig. S1 inthe supplemental material). An examination of the sampleswith conventional and immunofluorescence microscopyshowed that all observed cysts were stained with FITC-coupledantibodies. Since microscopic enumeration is generally consid-ered the accepted standard method, we defined this as the100% recovery value. The agreement ratio was defined as thequotient between the FCM count and the IFM count, dis-played as a percentage, i.e., Xa � (C� FCM/C� IFM) � 100, whereXa is the agreement ratio (%), C� FCM is the average FCM

count, and C� IFM is the average IFM count. This agreementquota was established for three different assays, namely, for 1ml spiked PBS, for 1 ml spiked PBS and subsequent immuno-magnetic separation, and for the complete procedure for de-tection of cysts in tap water and of naturally occurring cysts inwastewater (Table 1).

For the first two assays, Xa was around 96 to 98%, with a lowstandard deviation of �2%. For spiking of 1 liter of tap water,the recovery with the complete method was still high (102%),but the standard deviation was much higher (Table 1). False-positive results were never encountered for the negative con-trols of these experiments. The slightly higher FCM/IFM re-covery ratio for tap water might be due to particles in tap waterthat are not all eliminated during IMS and thus form a layer onthe filter area, eventually covering some cysts and impedingmicroscopic enumeration. This effect was even more pro-nounced with wastewater samples (Table 1).

In conclusion, flow cytometric enumeration seems to beequivalent to enumeration by fluorescence microscopy, andthus all control measurements for spiking experiments wereconducted by employing flow cytometry.

FIG. 1. FCM detection of FITC-labeled Giardia lamblia cysts in 1 ml of PBS. (Top left) Histogram of green fluorescence (520 nm) count. (Topmiddle) Histogram of red fluorescence (630 nm) count. (Top right) Histogram of SSC count. (Bottom left) Bivariate dot plot of green fluorescence(520 nm) versus SSC. (Bottom right) Dot plot of 520 nm versus 630 nm. Events are defined based on SSC, 520 nm, and 630 nm. The events ofgate R1 were restricted to the events inside gate RN1, establishing the multigate RN1-R1. SSC versus 520 nm and gate R2 are displayed becausethis distribution pattern and gating are often used in the literature as the only discrimination parameters.

TABLE 1. Quantification of Giardia lamblia cysts in PBS and tap water by either flow cytometry or fluorescencemicroscopy, using different assay procedures

Matrix ProcedureMean cyst count � SD

Xa (%)a

FCM IFM

1 ml spiked filtered PBS Detection only 16,248 � 163 16,782 � 273 96.8 � 1.95 ml spiked filtered PBS IMS and detection 16,057 � 202 16,413 � 179 97.8 � 1.61 liter spiked tap water Filtration, resuspension, IMS, and detection 12,348 � 824 12,159 � 966 101.6 � 10.5100 ml wastewater Filtration, resuspension, IMS, and detection 827 � 25 773 � 16 106.9 � 3.96

a Defined in Results. Values are means � normalized root mean square deviations (n � 3 experiments).

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(ii) Quantification limit for detection of cysts in tap water.We tested the method with spiking levels between 0 and 10,000cysts in order to determine the detection and quantificationlimits of the method (Fig. 2).

The average recovery of cysts from 1 liter of tap water, withturbidities ranging from 0.2 to 0.4 NTU, was 92.9% � 17.4%for all tested seeding levels. Spiking levels above 100 cysts perliter resulted in an average recovery of 97% � 8.4% (see TableS1 in the supplemental material).

Employing our adapted column-washing protocol, the FCMdot plots showed low background signals and clear separationof the cyst cluster from the background for both spiked PBS

and recovery from 1 liter of tap water (Fig. 3). Additionally,there were no false-positive results encountered for all of the1-liter tap water negative controls (Fig. 3c).

Different definitions for detection, determination, and quan-tification limits are used in validation documentation. In thiscase, the detection limit was defined as the spiking level atwhich 50% of the measurements were falsely negative, and thedetermination limit was defined as the spiking level at whichreliable detection involved a high statistical security (95%).The quantification limit corresponds to the seeding level atwhich a statistically safe quantification can be performed. Inorder to establish an estimated determination limit, we usedtwo different approaches for validation purposes (43). In spik-ing 10 to 20 cysts, we encountered one false-negative result(Fig. 2b; see Table S1 in the supplemental material), so weconsidered the mean value of 10 cysts (Fig. 2b, data point A)the detection limit due to the lack of more data points, and thisvalue was adopted for calculation purposes.

The first approach (46) for estimating the determinationlimit consisted of adding 3 times the standard deviation to thedetection limit, resulting in around 40 cysts per liter. Analo-gously, the quantification limit was established by adding 10times the standard deviation to the average detection limit,resulting in 110 cysts per liter.

The second approach (43) considered the determinationlimit to be the lowest value (Fig. 2b, data point B) lackingfalse-negative measurements where it was possible to statisti-cally significantly distinguish between positive signals and neg-ative control signals. For statistical significance, 2 times thestandard deviation of this determination limit may not inter-fere with 2 times the standard deviation of the negative controlor background noise. Based on this criterion, the determinationlimit was found to be 35 Giardia lamblia cysts per liter. In thisapproach, addition of 3 standard deviations to the determinationlimit leads to a quantification limit of 71 cysts per liter (43).

Combining both approaches, the estimated determinationlimit ranges from 35 to 40 cysts per liter, and the quantificationlimit ranges from 71 to 110 cysts per liter.

In our opinion, this conservative consideration may allowsafe quantification for water samples. Of course, this should beconfirmed experimentally for samples containing naturally oc-curring cysts, which may have lower binding affinities for thespecific surface antibodies. Reduced binding may be due toeither damaged or stressed cyst walls derived from either me-chanical or chemical stress encountered in water supply net-works (30) or from attachment to particles sterically hinderingantibody binding.

(iii) Recovery from 100 liters of tap water. Three trials wereconducted with the inline filter setup. The average spiking levelwas 140 � 30 cysts, resulting in an average recovery of 67% �5.13%. Figure 4 illustrates the increase in background signalfor this large water volume. Fortunately, the cysts were stillwell distinguishable from background signals.

(iv) Recovery from spiked pond and river water. Spikingexperiments with samples of different water quality were con-ducted in order to test the robustness of the proposed methodfor use with different matrices. Seeding levels for pond andriver water samples were in the range of 800 � 100 cysts perliter, representing 20 times the determination limit for valida-

FIG. 2. Calibration of spiked Giardia lamblia cysts by applying thecomplete method. (a) Number of spiked cysts versus number of recov-ered cysts from 1 liter of tap water. The full line indicates the linear-ization function y � 0.9293x 19.23 (r2 � 0.9998). (b) Zoomed-inversion of the 0- to 260-cyst count region of panel a. The full lineindicates the linearization function y � 1.0275x � 4.71 (r2 �0.9984), limited to the data points for up to 260 Giardia cysts perliter. Areas of the estimated detection, determination, and quantifi-cation limit ranges are marked. Point A is the highest seeding levelencountering a false-negative measurement. Point B is the lowest onenot encountering a false-negative measurement.

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tion purposes according to the instructions of the Swiss Ac-creditation Service (43).

The recovery from 1 liter of river water with a turbidity of 5to 6.5 NTU was 94.9% � 9.7%. Similar results were obtainedfor pond water (see Table S2 in the supplemental material). Asexpected, the background of such samples was much increasedcompared to that for tap water. No false-positive results wereobserved with the negative controls (Fig. 5).

Although the turbidity of the pond water was lower than that

of river water, the filtration process for the pond water wasmuch more difficult and time-consuming. A higher filtrationpressure was applied to the pond water samples (��5 � 103

Pa) because the filter clogged more rapidly. This higher pres-sure may have resulted in a lower recovery and a higher stan-dard deviation for the pond water samples (see Table S2 in thesupplemental material).

Application of the method to naturally contaminated waste-water samples. In order to test the method for detection ofnaturally occurring Giardia cysts and to determine its applica-bility to samples with higher turbidities, raw wastewater wasanalyzed by flow cytometry and fluorescence microscopy. Thesamples had turbidities ranging from 250 to 300 NTU. The cystconcentrations were 826.7 � 25.2 and 773.3 � 16.3 cysts per100 ml of sample by FCM and IFM, respectively, resulting in anXa value of 106.9% � 4% (Table 1). As already observed for tapwater, the number of detected cysts was higher when FCM wasused. This supports the hypothesis that particles cover and hidecysts on the filter, leading to reduced visible fluorescence and thusto false-negative results. When we spiked these samples, recoveryof 84% of the spiked cysts was achieved (Fig. 6).

DISCUSSION

Today’s standard immunofluorescence detection methodsbased on the USEPA 1623 method require skilled personneland are thus influenced by associated factors, for example,viewer fatigue (17). They are considered to be tedious, expen-sive, and only semiquantitative and often provide low and

FIG. 3. FCM detection of spiked Giardia lamblia cysts in buffer and tap water. Bivariate FCM dot plots (520 nm versus SSC and 520 nm versus630 nm) are shown. (a) One milliliter of PBS spiked with 200 Giardia cysts. (b) One liter of tap water spiked with 200 Giardia cysts, with the wholemethod applied (filtration/resuspension, IMS, and FCM). (c) One liter of tap water (negative control), with the whole method applied (filtration/resuspension, IMS, and FCM).

FIG. 4. Detection of spiked Giardia lamblia cysts in a 100-liter tapwater sample by FCM. Examples of bivariate FCM dot plots (520 nmversus SSC and 520 nm versus 630 nm) are shown for a 100-liter tapwater sample spiked with 150 Giardia cysts, with the whole methodapplied (filtration/resuspension, IMS, and FCM). An average of 140 �30 cysts were spiked, resulting in an average recovery of 67% � 5.13%.

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variable recoveries (6), usually ranging from 0.5% to 53% forenvironmental water samples (11).

Molecular approaches based on PCR can be highly sensitiveand specific down to a single cyst per sample if appropriateconcentration and purification are achieved. Nevertheless, de-tection is often hampered by PCR-inhibiting substances, andresults are often semiquantitative (40). Additionally, PCR islikely to be affected by nonspecific DNA contaminations orcarryovers from previously amplified DNA leading to false-positive results (25, 39).

Flow cytometry with fluorescence-activated cell sorting(FACS) is used increasingly as an alternative method for sep-aration and enumeration, but difficulties are encountered when

autofluorescent algae are present and cross-reactions of theMAbs lead to unspecific binding (2, 8, 48). Furthermore, ex-pensive and difficult-to-operate cell sorters are required, whichinhibits wide application. Some of these problems were ad-dressed by Ferrari and Veal (14), who developed a two-colordetection-only assay for Giardia cysts leading to no false-pos-itive results but also having a moderate recovery of 39% for aseeding level of 90 � 28 cysts in 10 liters of backwash water.

The method presented here consists of a prefiltration step inorder to eliminate large particles and contaminants. One-literwater samples are filtered through a 5-�m membrane. Thecysts are resuspended in 5 ml PBS and labeled with a specificFITC-coupled surface antibody. Subsequently, the Giardiacysts are separated immunomagnetically with commercial anti-FITC MACS MicroBeads with a diameter of 50 nm, resultingin a 1-ml sample analyzed by detection-only flow cytometry.This entire process requires only 100 min. Due to their smallsize and lack of autofluorescence, the beads do not need to beremoved from the microorganisms after IMS and can remainin the sample for enumeration. The MicroBeads do not createproblems that have been reported previously (9, 33), such ascyst detachment from large beads due to shear forces or inter-ference with immunofluorescence detection. Thus, it is unnec-essary to dissociate beads and cysts before detection, leading toa simple protocol. The short incubation time, increased recov-ery, and simple handling of the MACS technology were re-ported previously for Cryptosporidium detection (9).

Surprisingly, in contrast to the case for other detectionmethods relying on fluorescent dyes and flow cytometry (13),we did not encounter any false-positive results among the non-

FIG. 5. FCM detection of Giardia lamblia cysts spiked into 1-liter samples of different surface waters. Examples of positive and negativesamples for application of the complete method (filtration/resuspension, IMS, and FCM) are shown. Bivariate FCM dot plots (520 nm versus SSCand 520 nm versus 630 nm) are shown. (a) Pond water spiked with 800 cysts. (b) River water spiked with 800 cysts. (c) River water negative control.(d) Pond water negative control.

FIG. 6. Comparison of method recoveries for spiking with Giardialamblia cysts, depending on the matrix.

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spiked samples, and furthermore, potential cross-reactions ofthe antibodies did not obviously impair the demonstrated ro-bustness of the method. Furthermore, we achieved recoveriesexceeding 90% for all drinking water samples when we seeded75 cysts or more; a similar performance of our method wasobserved with environmental water samples, with time require-ments of less than 2 h. As shown in the comparison with IMFanalysis, flow cytometric enumeration seems to be less prone tosignal hiding, because during sample analysis, cysts and parti-cles are separated and not deposited together on a membranesurface.

Because all commercially available antibodies target thesame epitopes on Giardia sp. cysts (30), noncompetitive sur-face staining with two different dyes is not yet possible. Ac-cording to our results, this might not be necessary in order toobtain reliable results when monitoring for Giardia sp. con-tamination in drinking, surface, and wastewater.

The determination limit was estimated to be around 40 cystsper liter for drinking water, and thus the goal of detecting 1cyst per 10 to 100 liters of water (50) is not reached by theproposed method. Nevertheless, adaptation of the filtrationand resuspension process or an alternative preconcentrationmethod might enhance the performance. The first assays fil-tering 100 liters of tap water indicate that the method could becapable of robustly detecting Giardia cysts at 100-times-lowerconcentrations.

Due to the 200-�l volumetric sampling port of the FCMused in this study, the probability of deviation was muchhigher, as the flow cytometer actually analyzes 200 �l of the1-ml sample. This increased statistical deviation adds to theloss of cysts during the process, resulting in the observed de-viations. This becomes especially significant for seeding levelsbelow 100 cysts. It can be speculated that the use of an FCMequipped with a larger volumetric sampling port or a nonre-stricted sampling port, thus lowering the statistical deviationscompared to those with the cytometer used here, could alsoresult in a lower determination limit. Combining this with theabove-mentioned 100-liter sample preconcentration, the de-tection of 0.1 cyst per liter might be achieved, thus meeting thepreviously mentioned detection requirements. Nevertheless,this would be, in agreement with all available methods, still fartoo low to meet one theoretical maximum acceptable concen-tration of 1.7 � 10�2 cyst/1,000 liters (which would require theprocessing of at least 60,000 liters of water to detect a singlecyst [19]).

The drawbacks of the proposed method relative to PCRdetection might be overcome by employing peptide nucleicacid (PNA) probes, as proposed by Ferrari and Veal (14), forspecies-specific detection. Combining this method with otherstaining methods in order to distinguish physiological states,such as potential infectivity (e.g., propidium iodide live-deadstaining), would be an important improvement, and earlierreports indicated a good correlation with in vitro excystationprotocols for Cryptosporidium parvum (5).

Considering the application of our method to complex ma-trices, the wastewater experiment can be considered successful.Naturally occurring cysts were detected, despite the fact thatdetection can be difficult due to environmental influences onthe cyst wall (30). To date, we have not been able to elucidatethe impact of these effects, as both detection methods rely

on the same principle, i.e., antibody binding to the cellsurface.

In summary, this method has shown reliable results for var-ious matrices and has the potential to find wide applications,which are not limited to use with a flow cytometer only, as acombination with PCR or IFM detection is also possible. Thehigh recoveries (Fig. 6) for different matrices and the lowdependence on highly skilled laboratory personnel are addi-tional benefits. The convenient handling and about 100-mintime requirement appear very promising, and the costs permeasurement range at the moment from CHF 25 to 32 (US$28to 36 or EUR 20 to 25, based on exchange rates as of May2011) for consumables only. Finally, the applicability to variousmatrices and the potential to adapt the method to the detec-tion of virtually every microorganism show the opportunitiesrelated to this method.

ACKNOWLEDGMENTS

We thank the Federal Office for Civil Protection (project 350001621)and the Federal Office of Public Health (project 70106) for financialsupport of this study.

We thank the Spiez Laboratory, especially Nadia Schurch, for greatsupport.

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