2003 Annual Report | Development and Evaluation of Methods for the Concentration, Separation, Detection, and Viability/Infectivity of Three Protozoa from Large Volume of Water| Research Project Database | NCER | ORD

2003 Progress Report: Development and Evaluation of Methods for the Concentration, Separation, Detection, and Viability/Infectivity of Three Protozoa from Large Volume of Water

EPA Grant Number: R828043
Title: Development and Evaluation of Methods for the Concentration, Separation, Detection, and Viability/Infectivity of Three Protozoa from Large Volume of Water
Investigators: Tzipori, Saul , Sheoran, Abhineet , Widmer, Giovanni , Zuckermann, Udi
Current Investigators: Tzipori, Saul , Buckholt, Michael , Widmer, Giovanni , Zuckermann, Udi
Institution: Tufts University , Mekorot Water Supply Company Ltd
Current Institution: Tufts University
EPA Project Officer: Nolt-Helms, Cynthia
Project Period: March 1, 2000 through March 1, 2003
Project Period Covered by this Report: March 1, 2003 through March 1, 2004
Project Amount: $525,000
RFA: Drinking Water (1999)
Research Category: Drinking Water

Description:

Objective:

The objective of this research project is to respond to the U.S. Environmental Protection Agency’s (EPA) requests for improvements in the concentration and separation of Cryptosporidium and Giardia from ambient water samples. Dr. Tzipori of Tufts University submitted a proposal to EPA to adapt and further investigate the utility of a Latham bowl and portable continuous flow centrifugation (PCFC). The disposable Latham bowl is a medical device developed by Haemonetics that uses continuous flow centrifugation toeparate blood cells.

At Tufts University School of Veterinary Medicine, North Grafton, Massachusetts, a portable device was developed and assembled from a stationary differential continuous flow centrifuge usually employed for blood cell separation (Haemonetics). The unit was employed for the purpose of concentrating Cryptosporidium and Giardia from large volumes of water. Following deposition onto the wall of the disposable plastic centrifuge bowl, the oocysts and cysts are dislodged by injection of 5 mL of buffer and vigorous shaking to yield a final elution buffer concentration of 1 percent sodium dodecyl sulphate with 0.01 percent Tween 80 and 0.001 percent antifoam A. Following a second centrifugation and aspiration of the supernatant, the organisms are separated from the pellet by ion mobility spectrometry (IMS) and reacted with specific fluorescein isothiocyanate-conjugated monoclonal antibodies and enumerated via fluorescence microscopy. The entire procedure requires about 2 hours.

The following alternate test procedure (ATP) study addresses the modification of the sample concentration procedure in Method 1623: Cryptosporidium and Giardia in Water by Filtration/IMS/FA, U.S. EPA-821-R-01-025, Office of Water, April 2001. The modified method proposed by this ATP replaces filtration and employs a PCFC technology.

Progress Summary:

Study Design

Laboratories. Two types of laboratories were required for the study: a flow-cytometry laboratory to prepare and distribute the spike samples (Wisconsin State Laboratory of Hygiene [WSLH]) and four analytical laboratories approved by EPA to perform Cryptosporidium and Giardia testing in water samples (Clancy Environmental Consultants [laboratory #1 in this report]; Analytical Services, Inc. [laboratory #2]; Environmental Associates [laboratory #3]; and the Upper Mohawk Regional Water Board [laboratory #4]).

Sample Matrices. Each analytical laboratory processed a total of eight samples: four initial precision and recovery (IPR) samples, one unspiked reagent water (method blank), two matrix spike (MS) samples (MS/matrix spike duplicate [MSD]), and one unspiked matrix sample.

Each participating laboratory was required to provide its own reagent water and raw surface water. Raw surface water samples were collected as grab samples from a local surface water body serving as a source for drinking water. One of the participating laboratories was unable to obtain its own surface water sample so a sample was provided from the Utica area of New York.

Spike Samples Preparation and Distribution. The flow-cytometry laboratory prepared six spiking suspensions with live organisms for each participating laboratory and one blank suspension.

A target value of 150 oocysts and cysts was established for the spiking suspensions used in the study. The target value was unknown to the participating laboratories. The organisms were enumerated by the flow-cytometry laboratory according to sections 11.2-11.2.6 in Method 1623. Each spike sample was placed in a marked tube. The referee laboratory distributed the spiking suspensions to the participating laboratories as a priority overnight delivery. The sample tubes were packed in compliance with Centers for Disease Control and Prevention etiologic agent shipping regulations.

Spiking Procedures. Oocysts and cysts were spiked into a 50 L carboy according to the procedure described in Method 1623, sections 11.4-11.4.3.1.6. The carboy was placed on a stir plate and continuously mixed using a stir bar.

Study Implementation

Technical Approach: Tier Level . This round-robin validation study was designed to meet the EPA performance-based measurement system (PBMS) Tier 2 method validation requirements for method modification for a nationwide approval for ambient (surface) water (see Table 1).

Table 1. Comparison of PBMS Tier 2 Validation Requirements and the
Components of This Validation Study

PBMS Tier 2 Requirements	Components of the Study
Three Participating Laboratories	Four Participating Laboratories
12 IPR Samples	16 IPR Samples (Four Replicates of Spiked Reagent Water in Four Laboratories)
Three Method Blanks (Reagent Water)	Four Blanks (One in Four Laboratories)
Six MS/MSD Samples + Three Unspiked MS	Eight MS/MSD Samples (One MS and One MSD in Four Laboratories) + Four Unspiked Matrix Samples (One Unspiked Matrix Sample Per Laboratory)

Sample Matrices. Two sample matrix types were used in this study: reagent water and surface water. Each participating laboratory provided reagent water and raw surface water collected as a grab sample from a local reservoir/lake with the exception of one laboratory, which could not get its own source water, so source water samples from the Utica area (Rome reservoir source water) were used. One of these ambient water sources was a lake, and the collected water sample had a turbidity result of greater than 10 NTU.

Sample Preparation and Distribution. A flow-cytometry laboratory (WSLH) conducted the sample preparation and distribution to the participating laboratories. The laboratory calibrated its flow cytometer to ensure the accurate sorting of the oocysts and cysts per section 11.2 in the April 2001 version of Method 1623.

The flow-cytometry laboratory used its routine source of oocysts and cysts and supplied the study coordinator with all of the information pertaining to the origin and purification process. Spiking suspensions were prepared using Cryptosporidiumparvum oocysts (Sterling Parasitology, Harley Moon, collected on 4/15/03) less than 3 months old and Giardiaintestinalis cysts (Waterborne, H3, collected on 4/23/2003) less than 2 weeks old. The spike target was 150 oocysts/cysts. The spike levels were blind to the participating laboratories. The laboratory supplied of all the information regarding ongoing sample calibration to Tufts University. The information then was submitted to EPA.

Distribution of Spiking Suspensions to the Participating Laboratories. After generating all spiking suspensions using the flow cytometer, the flow-cytometry laboratory prepared four tubes containing water and Tween but no organisms (the blind unspiked tubes that were sent to the laboratories).

Sample Spiking Procedures. The spiking suspension was vortexed for a minimum of 2 minutes. A volume of 500 µL diluted antifoam (80% Antifoam A, 20% reagent water) was added, and the whole volume was vortexed for 2 minutes. The spike then was poured into the 50 L carboy. The tube was rinsed twice by adding 20 mL of reagent water that then was added to the empty tube. The tube was capped and vortexed for 2 minutes, and the rinsate was poured into the carboy. The spike suspension was mixed using a stir bar and stir plate for approximately 1 minute in the carboy.

Experimental Format of the Validation Study. A total of eight samples were processed in each participating laboratory (four initial precision and recovery samples, one method blank sample, two MS samples, and one unspiked matrix sample). The samples were pumped into the inlet port of the PCFC at a flow rate of 0.7 L per minute.

Following the concentration process, the centrifuge bowl was disassembled and the elution protocol was performed. After the elution process, the bowl contents of 250 mL were poured into a 250 mL centrifuge tube.

Laboratory procedures specified in the April 2001 version of Method 1623 were followed from section 13 onward. Dynal IMS and the Waterborne Aqua-Glo staining kit were used by each laboratory. Laboratory #1 used Merifluor slides, laboratories #3 and #4 used Super Stick slides, and laboratory #2 used Dynal Spot-On slides.

The laboratories processed samples that generated more than 0.5 mL of packed pellet after centrifugation according to sections 13.2.3 and 12.3.4 of Method 1623 . At laboratory #1, the acid disassociation fluid for all subsamples analyzed for each MS was placed onto one well. The Merifluor slide has a larger diameter than the Dynal Spot-On slides (the Dynal has a 14 mm diameter) and can accommodate 200 µL volumes, provided that the analyst takes extreme care when applying the sample to the well. The appropriate amount of 1.0 N NaOH was added to each well to neutralize the acid adequately.

Results

The results of the study are presented in Tables 2 through 7.

Table 2.Cryptosporidium and Giardia Spike Dose Using Flow Cytometry at the WSLH

Cryptosporidium Oocysts		Giardia Cysts
Initial Calibration	Ongoing Calibration	Initial Calibration	Ongoing Calibration
152	150	151	151
151	152	148	149
152	149	152	148
150	148	148	147
148	148	144	149
150	149	146	150
150		147
152		154
149		149
149		154
Mean: 150.3	Mean: 149.3	Mean: 149.3	Mean: 149
SD: 1.41	SD: 1.5	SD: 3.36	SD: 1.4
RSD : 0.93	RSD: 1.0	RSD: 2.25	RSD: 0.93

Table 3. Raw IPR and MS/MSD Data for Cryptosporidium and Giardia: Clancy Environmental Consultants, Inc., Vermont

Sample	Turbidity (NTU)/ Water Type	Packed Pellet (Volume)	Mean Spike Dose	No. of Recovered Organisms	Recovery (%)	Mean Recovery (%)	RSD/ RPD
*Cryptosporidium*
IPR1	NA	Trace	149.3	53	35.5	-	-
IPR2	NA	Trace	149.3	65	43.54	-	-
IPR3	NA	Trace	149.3	70	46.89	-	-
IPR4 (Method Blank)	NA	Trace	Unspiked	0	-	-	-
IPR5	NA	Trace	149.3	62	41.53	-	-
IPR	-	-	-	-	-	42	11
MS1	13.1 (Lake)	1.0	149.3	30	20.09	-	-
MS2	11.0 (Lake)	1.0	149.3	25	16.74	-	-
MS3	10.4 (Lake)	1.0	Unspiked	0	-	-	-
MS	-	-	-	-	-	18	13
*Giardia*
IPR1	NA	Trace	149.0	66	44.30	-	-
IPR2	NA	Trace	149.0	74	49.66	-	-
IPR3	NA	Trace	149.0	74	49.66	-	-
IPR4 (Method Blank)	NA	Trace	Unspiked	0	-	-	-
IPR5	NA	Trace	149.0	87	58.39	-	-
IPR	-	-	-	-	-	50	12
MS1	13.1 (Lake)	1.0	149.0	22	14.77	-	-
MS2	11.0 (Lake)	1.0	149.0	20	13.42	-	-
MS3	10.4 (Lake)	1.0	Unspiked	0	-	-	-
MS	-	-	-	-	-	14	7

Table 4. Raw IPR and MS/MSD Data for Cryptosporidium and Giardia: Analytical Services, Inc., Vermont

Sample	Turbidity (NTU)/ Water Type	Packed Pellet (Volume)	Mean Spike Dose	No. of Recovered Organisms	Recovery (%)	Mean Recovery (%)	RSD/ RPD
*Cryptosporidium*
IPR1	NA	Trace	149.3	21	14.07	-	-
IPR2	NA	Trace	149.3	46	30.81	-	-
IPR3	NA	Trace	149.3	42	28.13	-	-
IPR4 (Method Blank)	NA	Trace	Unspiked	0	-	-	-
IPR5	NA	Trace	149.3	39	26.12	-	-
IPR						25	30
MS1	0.9 (Lake)	1.5	149.3	82	54.92	-	-
MS2	0.9 (Lake)	1.5	149.3	75	50.23	-	-
MS3	0.9 (Lake)	1.5	Unspiked	0	-	-	-
MS	-	-	-	-	-	53	6
*Giardia*
IPR1	NA	Trace	149.0	65	43.62	-	-
IPR2	NA	Trace	149.0	88	59.06	-	-
IPR3	NA	Trace	149.0	63	42.28	-	-
IPR4 (Method Blank)	NA	Trace	Unspiked	0	-	-	-
IPR5	NA	Trace	149.0	61	40.94	-	-
IPR	-	-	-	-	-	46	18
MS1	0.9 (Lake)	1.5	149.0	80	53.69	-	-
MS2	0.9 (Lake)	1.5	149.0	92	61.74	-	-
MS3	0.9 (Lake)	1.5	Unspiked	0	-	-	-
MS	-	-	-	-	-	58	10

Table 5. Raw IPR and MS/MSD Data for Cryptosporidium and Giardia: Upper Mohawk Water Utility, Utica, New York

Sample	Turbidity (NTU)/ Water Type	Packed Pellet (Volume)	Mean Spike Dose	No. of Recovered Organisms	Recovery (%)	Mean Recovery (%)	RSD/ RPD
*Cryptosporidium*
IPR1	NA	0.1	149.3	79	52.91	-	-
IPR2	NA	0.1	149.3	97	64.97	-	-
IPR3	NA	0.1	149.3	94	62.96	-	-
IPR4 (Method Blank)	NA	0.1	Unspiked	0	-	-	-
IPR5	NA	0.1	149.3	108	72.33
IPR	-	-	-	-	-	63	13
MS1	1.83 (Hinckley Reservoir)	0.9	149.3	98	64.30	-	-
MS2	1.83 (Hinckley Reservoir)	0.9	149.3	127	83.72	-	-
MS3	1.83 (Hinckley Reservoir)	0.9	Unspiked	2	-	-	-
MS	-	-	-	-	-	74	19
*Giardia*
IPR1	NA	0.1	149.0	74	49.66	-	-
IPR2	NA	0.1	149.0	58	38.93	-	-
IPR3	NA	0.1	149.0	74	49.66	-	-
IPR4 (Method Blank)	NA	0.1	Unspiked	0	-	-	-
IPR5	NA	0.1	149.0	92	61.74	-	-
IPR	-	-	-	-	-	50	19
MS1	1.83 (Hinckley Reservoir)	0.9	149.0	78	50.34	-	-
MS2	1.83 (Hinckley Reservoir)	0.9	149.0	75	48.32	-	-
MS3	1.83 (Hinckley Reservoir)	0.9	Unspiked	3	-	-	-
MS	-	-	-	-	-	49	3

Table 6. Raw IPR and MS/MSD Data for Cryptosporidium and Giardia: Environmental Associates, Ltd., Ithaca, New York

Sample	Turbidity (NTU)/ Water Type	Packed Pellet (Volume)	Mean Spike Dose	No. of Recovered Organisms	Recovery (%)	Mean Recovery (%)	RSD/ RPD
*Cryptosporidium*
IPR1	NA	< 0.1	149.3	15	10.05	-	-
IPR2	NA	< 0.1	149.3	75	50.23	-	-
IPR3	NA	< 0.1	149.3	75	50.23	-	-
IPR4 (Method Blank)	NA	< 0.1	Unspiked	0	-	-	-
IPR5	NA	< 0.1	149.3	73	48.90	-	-
IPR	-	-	-	-	-	40	43
MS1	1.31 (Rome Reservoir)	0.5	149.3	6	4.02	-	-
MS2	1.24 (Rome Reservoir)	0.5	149.3	8	5.36	-	-
MS3	1.36 (Rome Reservoir)	0.5	Unspiked	0	-	-	-
MS	-	-	-	-	-	5	14
*Giardia*
IPR1	NA	< 0.1	149.0	35	23.49	-	-
IPR2	NA	< 0.1	149.0	91	61.07	-	-
IPR3	NA	< 0.1	149.0	77	51.68	-	-
IPR4 (Method Blank)	NA	< 0.1	Unspiked	0		-	-
IPR5	NA	< 0.1	149.0	47	31.54	-	-
IPR						42	36
MS1	1.31 (Rome Reservoir)	0.5	149.0	8	5.37	-	-
MS2	1.24 (Rome Reservoir)	0.5	149.0	19	12.75	-	-
MS3	1.36 (Rome Reservoir)	0.5	Unspiked	0	-	-	-
MS	-	-	-	-	-	9	41

Table 7. A Summary of Results Comparing Method 1623 Acceptance Criteria, PCFC Validation Study Mean Recoveries, and Individual Laboratory Performance Data

Matrix/ Organism	Method 1623 Acceptable Mean Recovery (%)	Method 1623 Acceptable Precision	CEC Mean Recovery (%)	RSD/RPD	ASI Mean Recovery (%)	RSD/ RPD	MVWA Mean Recovery (%)	RSD/ RPD	EAL Mean Recovery (%)	RSD/ RPD
Reagent Water Crypto-sporidium	21-100	55	42	11	25	30	63	13	40	43
Giardia	17-100	49	50	12	46	18	50.0	19	42	36
Method Blank Crypto-sporidium	-	-	-	-	-	-	-	-	-	-
Giardia	-	-	-	-	-	-	-	-	-	-
Source Water Crypto-sporidium	13-111	61	18	13	53	6	74	19	5	14
Giardia	15-118	30	14	7	58	10	49	3	9	41

Data Analysis and Discussion

Study results presented in Table 7 demonstrate that the PCFC meets and exceeds the acceptance criteria for Method 1623 and is capable of concentrating Cryptosporidium oocysts from 50 L of source water. Because laboratory #1 failed to get acceptable recoveries for Giardia spiked into turbid source water, the study could not demonstrate that each laboratory in the study could achieve acceptable recoveries in each of the matrices tested for each organism. The mean recovery in laboratory #1 for Giardia in source water was less than 1 percent under the minimum accepted value of 15 percent. One explanation for this result could be the interaction of the cysts with the material suspended within 50 L of more than 10 NTU source matrix during the process, which could lead to their degradation. Previous testing of the PCFC with waters having greater turbidities demonstrated much higher recoveries.

Laboratory #4 failed to get acceptable results for IPR1, MS1, and MS2. During the training, this laboratory demonstrated the ability to perform acceptable results when using the PCFC. The cause for the poor results during the validation was not identified; however, a potentially significant confounding variable was identified. During the training, the laboratory performed the wrist shaking by using its own older model wrist shaker. Because this model did not enable setting the desired rpm and orienting the clamps, Tufts University supplied a current wrist shaker model. Because of the expiration date of the spike samples, the laboratory did not have time to practice with the new wrist shaker. These factors suggest that a likely explanation for the low recoveries of the source water samples was insufficient elution of the oocysts and cysts from the bowl.

Future Activities:

The investigators did not report any future activities.

Journal Articles:

No journal articles submitted with this report: View all 1 publications for this project

Supplemental Keywords:

Cryptosporidium, Microsporidia, Enterocytozoonbieneusi, Encephalitozoon intestinalis, Giardia, protozoa, water, public water systems, , Ecosystem Protection/Environmental Exposure & Risk, Water, Scientific Discipline, Health, RFA, PHYSICAL ASPECTS, Drinking Water, Risk Assessments, Health Risk Assessment, Physical Processes, Monitoring/Modeling, bacteria, cryptosporidium, encephalitozoon, exposure and effects, microbiological organisms, pathogenic protozoa, microorganism, analytical methods, Giardia, human health risk, monitoring, detection, measurement, microsporidia, infectivity assays, public water systems, waterborne disease, cryptosporidium , microbial risk management, exposure, infectivity, microbial contamination, microbial monitoring, assessment technology, concentration device, assays, infective dose, measurement

Progress and Final Reports:
2000 Progress Report
2001 Progress Report
2002 Progress Report
Original Abstract
Final Report

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2003 Progress Report: Development and Evaluation of Methods for the Concentration, Separation, Detection, and Viability/Infectivity of Three Protozoa from Large Volume of Water

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