Analysis of Envirochek®Sampling Capsule versus Traditional String Wound Cartridges for Collection and Recovery of Cryptosporidium

The disease Cryptosporidiosis has affected the human population on six continents(1,2).

Cryptosporidiosis is caused by the protozoan Cryptosporidium, which until 1976 had been recognized as a pathogen only in calves and was not known to infect humans(5,11). Since then, reports of outbreaks have risen steadily. The most well-known outbreak in the U.S. occurred in Milwaukee, Wisconsin, in 1993, affecting 400,000 people and killing 110(10). Cryptosporidium outbreaks like the Milwaukee incident have increased the concern over waterborne transmission of such pathogens, and have brought to the forefront a focus on improved detection methods.

Cryptosporidium is a naturally occurring organism found in the environment; thus, eradication is unlikely. Therefore, the advancement of recovery and detection methods is highly critical. One of the first studies that evaluated the recovery efficiency of the most common sampling method using string wound filters demonstrated that only 2 to 8% of Cryptosporidium oocysts were recovered from the spiked sample(3). To improve the recovery efficiency of Cryptosporidium oocysts, Pall developed the Envirochek sampling capsule which has demonstrated recoveries exceeding 80%. This exceptional recovery rate, combined with a sampling process that is easier, more efficient, and safer to use than traditional methods, makes the Envirochek capsule a preferable method for collecting and recovering Cryptosporidium.

Accuracy, ease of use, and time savings are all important factors in the recovery of Cryptosporidium. The traditional recovery method, which specifies a string wound cartridge and a bulky filter housing, is difficult to assemble, requires at least 240 minutes to prepare for identification, and in multiple studies typically yielded recoveries of 2 to 54% (see details below). In comparison, the Envirochek capsule and protocol requires no pre-assembly, only 70 minutes to prepare for identification, and recovers greater than 80% of the oocysts.

Summary of Results

	String Wound Cartridge	Envirochek Capsule
Capsule Accuracy/Recovery Clancy Study II* (4)	10-23%	91%
Accuracy/Recovery Clancy Study III	54%	82%
Accuracy/Recovery Clancy Study 1 (3)	2-8%	Product not available
Collection Process Time (4 samples)	240 minutes	70 minutes
Filter Assembly	Required	Not Required
Disinfection	Required	Not Required
Stomaching	Required	Not Required

*A study performed by Clancy Environmental Consultants, St. Albans, VT, USA, compares the recovery efficiency of the Envirochek capsule to a 1 µm nominal string wound cartridge filter that is often used in the current Information Collection Rule (ICR) methodology. The methodology and results of this study
are presented below.

A study was conducted by Clancy Environmental Consultants to investigate the Cryptosporidium sampling and recovery process using the Envirochek capsule and a string wound filter. Below is a description of the test methods used to evaluate the two filters, as well as a comparison of the results obtained when the two methods were compared for accuracy, time savings in the recovery steps, ease of use, and increasing laboratory personnel safety.

Two Envirochek capsules and two string wound cartridges in 10-inch housings were attached to a five-leg manifold. A 142 mm, 3 µm polycarbonate track-etch membrane capture filter was attached to the fifth leg of the manifold and used as a positive control. An injection port was used to simulate raw water turbidities by injecting concentrated natural water particles. A material equivalent of 1 NTU water was prepared from in-house environmental sample concentrates. This suspension was then injected continuously over the duration of the test by a metering diaphragm pump. Following the initial wetting of the filters, a flow rate of 2 L/min was maintained for each test filter and the control filter. A challenge spike targeting 6.0 x 10³ Cryptosporidium  oocysts was injected by a second metering diaphragm pump through an injection port into the feed line common to all test filters and the control filter. The testing period lasted five minutes for a total volume of 10 liters per filter. The filters were then removed for extraction and analysis using the manufacturer's recommended procedures. The process was repeated with two Envirochek capsules and two string wound cartridges.  

Upon completion of the testing, a thorough comparison of the two filtration methods was completed, from the sampling process through the recovery of the organism. Below is an overview of the major differences observed between the Envirochek capsule and the string wound filter.

Accuracy and Consistency

Accuracy is the most important factor in evaluating a product for Cryptosporidium recovery. In this study, the Envirochek capsule showed significantly higher recoveries of the challenge Cryptosporidium oocysts than the string wound cartridges. Table 1 illustrates the results.

Table 1
Recovery Comparison

	Envirochek Capsule	String Wound Cartridge
Sample Identification	1  2  3  4	1  2  3  4
Recovery	66 77 84 100	54 28 58 78
Average Percent Recovery	82	54*
Standard Deviation	± 12	± 18

*While the Envirochek capsule had recoveries over 50% higher than the string wound filter, even this data may be an optimistic view of the performance of the string wound filter, as two previous studies showed recoveries as low as 2% and no higher than 23%(3, 4).

One potential reason for this difference in Cryptosporidium recovery could be the eluate volume collected off the filters. Typically, it is preferable to have a minimum eluate volume in order to minimize loss of oocysts and reduce subsequent processing steps. Large eluate volumes that require centrifugation for concentration have been shown to be a contributing factor to lower oocyst recoveries. The Envirochek capsule results in approximately 240 mL of eluate upon completion of the collection process, while the current method of washing a string wound filter results in approximately 3,500 mL of eluate that must be centrifuged down to a pellet for analysis.

Time Comparison in the Recovery Steps

Using the string wound cartridge, the recovery process for eluting the oocysts from the filter media after sample collection was very time consuming and tedious. The user must cut apart the cartridge and wash the filter material to recover the organisms. With the Envirochek capsule, the user simply pours the elution buffer directly into the capsule and agitates it on a laboratory shaker. The time required to complete the sampling process using an Envirochek capsule is significantly less than the time required to process a string wound cartridge (see Table 2). In the Clancy Environmental Consultants study, four Envirochek capsules could be processed to the point of detection in a total of 70 minutes, while the string wound cartridge required 240 minutes of processing time. Overall, the Envirochek capsule process saves the user 170 minutes of processing time. This time savings can be increased significantly by processing up to eight Envirochek capsule samples at once on a laboratory shaker.

Table 2
Process Time* Evaluation for Four Envirochek Capsules and Four String Wound Cartridges

Process Step	Envirochek Capsule	String Wound Cartridge
Filter Cutting	0	60
Shaking	40	0
Stomaching	0	120
Labware Cleaning	30	30
Filter Housing Cleaning	0	30
Total Time (minutes)	70	240

*Time represents elapsed time (in minutes) from completion of filtration to beginning of slide preparation for the detection phase.

Ease of Use in the Sampling Process

When testing in the field, the Envirochek capsule replaces the string wound cartridge and housing assembly. This provides several advantages to the user. The Envirochek capsule is a self-contained filtration unit that includes a housing and integral filter element, thus there is no assembly required(8). Consequently, housing disinfection is not required(8). Because Envirochek capsules are disposable and can be easily replaced, samples can be collected quicker, more samples can be collected in the same amount of time, and the risk of cross-contamination from previous samples is eliminated. When sample collection is complete, the user simply places the green vinyl end caps on the inlet and outlet of the capsule and transports it on ice to the laboratory for analysis.

Laboratory Personnel Safety

The process of using the Envirochek capsule for oocyst recovery is safer for laboratory personnel because it does not require direct handling of the contaminated filter media. When the sample is received by the laboratory, a buffer is added to the encapsulated Envirochek capsule to elute the oocysts. Because elution takes place within the capsule housing, the potential exposure of the technician to oocysts is eliminated. After the elution buffer is added, the Envirochek capsule is placed in a laboratory shaker to dislodge the oocysts from the membrane. This procedure differs from the string wound cartridge procedure, which requires the cartridge to be cut open with a razor blade and washed in a buffer solution. The washing process, also known as stomaching, looks similar to how laundry was scrubbed on washboards in the 1800s, and can potentially expose technicians to oocysts(8).

Throughput with Turbid Water

High turbidity typically suggests contamination in water sources. Water systems are most vulnerable to microbial risk when they exceed a turbidity reading of 5 NTUs(8). Filtering turbid waters can be a difficult and time-consuming process. In a study conducted by Pall, data was collected on the Envirochek capsule using water ranging from 5-100 NTUs to demonstrate the predetermined amounts of clay dust in the water. The water was then pumped through the filter (flow rate) and the volume collected at specific time intervals was measured. 140 liters was chosen as an endpoint for gathering the data.

As Table 3 illustrates, 140 liters of water was able to pass through the Envirochek capsule at 5, 20, and 60 NTUs. At higher turbidities, the contamination significantly affects the filter throughput but still allows the collection of over 30 liters at a turbidity of 100 NTU.

Table 3
Volume vs. Water Turbidity*

U.S. EPA methods 1622 and 1623, which list the Envirochek capsule as an approved technique, only require 10 liters of water to be sampled. In the Pall study, 10 liters of water was able to pass through the Envirochek capsule at all of the above-mentioned turbidities in a reasonable amount of time. As Table 4 illustrates, it takes 11 minutes or less to filter 10 liters of water, with water ranging in turbidities from 5 to 100 NTUs. Limited testing also has shown that 10 liters of water at 1,000 NTUs will pass through the Envirochek capsule in approximately 30 minutes.

Table 4
Time vs. Water Turbidity

**All charts are an indication of typical performance. Turbidity was controlled using clay dust.

In this study, the Envirochek capsule demonstrated higher recoveries in various matrix conditions, the ability to filter 10 liters of water in highly turbid conditions, and the best overall performance when compared to one type of string wound cartridge traditionally used for Cryptosporidium testing. The time that is required for sample processing was significantly reduced, and a much lower eluate volume was produced. This reduced the time required for further processing steps, and increased recovery accuracy.

Studies show that Cryptosporidium is found in 65 to 97% of surface waters in the United States (lakes, streams, and rivers)(9, 12). Work is also occurring around the world to determine the safety of public drinking and recreational water. The technology used in the past has been inconsistent and provided highly variable results. Pall's Envirochek sampling capsule provides technicians in both the public and private sectors the opportunity to have confidence in their results and help to protect the public from Cryptosporidium
contamination. The Envirochek capsule provides an easier, quicker, and safer way to recover Cryptosporidium oocysts. And, with recovery levels of greater than 80%, it is easy to conclude that the Envirochek sampling capsule from Pall is a preferable method for the collection and recovery of Cryptosporidium.

1. Badenoch, J. et.al. 1990. Cryptosporidium in Water Supplies. Dept. of the Environment and Dept. of Health. London.
2. Black, R.E. January-February 1990. Epidemiology of Travelers' Diarrhea and Relative Importance of Various Pathogens. Reviews of Infectious Diseases. 12, Supplement 1.
3. Clancy, J.L.; Gollintz, W.D. and Tabib, Z. (1994) Commercial Labs: How accurate are they? Journal of the American Water Works Association 86, 89-97.
4. Clancy, J.L., T.M. Hargy, and S. Schaub. 1997A. Evaluation of protozoa sampling and processing methods for near term monitoring requirements. USEPA Contract No. 68-C5-3909.
5. City & County of San Francisco, San Francisco Public Utilities. Cryptosporidium White Paper.
6. Fayer, R., et.al. 1993. General Biology of Cryptosporidium, in Cryptosporidiosis of Man and Animals. Edited by Dubey, J.P.; Speer, C.A.; and Faber, R.
7. Federal Register Vol. 54, No. 124, 27502.
8. Federal Register Vol. 59, No. 128, 6416-20.
9. Lechevallier MW, Norton WD, and RG Lee. Occurrence of Giardia and Cryptosporidium spp. in surface water supplies. Applied Environmental Microbiology 1991;57(9):2610-6.
10. Nannis, P.W., Commissioner of Health, City of Milwaukee. April 19, 1993. Testimony to the Subcommittee on the Health and Environment.
11. Pennsylvania Department of Environmental Protection, Bureau of Water Supply and Community Health, Division of Drinking Water Management and Pennsylvania Department of Health, Bureau of Epidemiology, Division of Communicable Disease Epidemiology (April 1996). Cryptosporidium Action Plan.
12. Rose, J.B. Occurrence and Significance of Cryptosporidium in Water. Journal of the American Water Works Association 1988;80(2): 53-8.

Envirochek and Envirochek HV Sampling Capsules