Focus
I
ntense research efforts are currently underway to develop new analytical methods for detecting the waterborne protozoa, Cryptosporidium and Giardia. Since the 1993 Cryptosporidium outbreak in Milwaukee, which is believed to have caused 104 deaths and sickened an estimated 400,000 people, the U.S. Environmental Protection Agency (EPA) has been under pressure to regulate protozoan parasites in drinking water The biggest challenge that EPA has faced in regulating protozoa in water has been in developing a suitable method for detecting
would be required to monitor for Cryptosporidium under the information collection rule (ICR). However, the ICR method for detecting Cryptosporidium and Giardia—based on an indirect fluorescent antibody (IFA) procedure—has been scrutinized by many and found to be plagued with problems, including low recovery efficiencies lack of reproducibility low sensitivity and the inability to determine viability and speciation In addition the procedures are difficult and the interpretation of results requires a highly trained skilled microscopist
NEW METHODS
EPA says it recognizes the limitations of the ICR method and is in the process of revising it. A draft of a modified method (referred to as EPA Method 1622) is being evaluated and revised based on the results of a series of roundrobin evaluations, which began in November by EPA-solicited commercial labs. EPA will not release the details regarding Method 1622 until the roundrobin evaluations are completed. Method 1622 is expected to be released later this month
FOR DETECTING
Crvhtnsbnridium and Cinrdin (Awfll Chpw .QQ1^ Pt7 7*31 A"i
Cryptosporidium exists in natural waters as ~!>um sized oocysts, surrounded by a tough coating that makes them resistant to standard disinfection procedures. Filtration steps to reduce the turbidity of treated water are therefore the primary lines of defense against the micrometer-sized pathogen in most public water systems. When water filtration systems fail however serious public health problems can ensue In humans ingestion of Cryptosporidium oocysts leads to infection of the small intestine with diarrheal symptoms that tvoically last for one to two weeks For
CRYPTOSPORIDIUM
EPA seeks alternative to indirect fluorescent antibody procedure.
those individuals with weakened immune
systems, infection is much more serious and is often life-threatening. The regulatory side In a May 1996 press release, EPA announced that water utilities serving >10,000 people
According to William Telliard of EPA's Office of Water, "Method 1622 has been drafted along the lines of a performancebased measurement system (PBMS)" (Anal. Chem. 1996, 68, 733 A; Anal.
Analytical Chemistry News & Features, January 1, 1998 4 9 A
Focus
Analytical methods With Cryptosporidium testing, large water volumes (10-1000 L) must be processed because of the low concentrations of oocysts typically found in raw and, particularly, treated waters. Regardless of the method chosen, some sort of concentration step must therefore be included. The IFA method for detecting Cryptosporidium, which is based on a proposed American Society for Testing and Materials (ASTM) method, involves passing up to 100 L of raw water or 1000 L of finished water through cartridge filters (made of yarnwound polypropylene). The retained particles are eluted from the filters and the washings are centrifuged to further concentrate the oocysts The sample is purified by a density flotation procedure in which unwanted debris sinks to the bottom
Chem. .997, 69, ,16 A), with various options that may be amended, if certain specifications are met. By allowing flexibility in the method, alternative procedures can be used, he said, provided that they satisfy data quality objectives—requirements for precision, bias, sensitivity, and the range of conditions over which satisfactory data can be obtained. Eventually the agency expects to require the use of drinking-water reference materials standards for which criteria will be established by the National Institute of Standards and Technology (NIST) to test the accuracy of a given method The mechanism for incorporating standard reference materials into the reeulatory framework for Crvbtosboridium monitoring has not been worked out "We're not there yet" says Telliard The first use of Method 1622 will be to conduct a survey, which includes voluntary participation by the utilities and is scheduled to begin this month. The survey will complement ICR data and assist EPA in conducting regulatory impact analysis for controlling Cryptosporidium ana other pathogens. Results from the ICR survey are expected to determine how often and to what degree Cryptosporidium ana other microbes occur in the source waters of the nation's drinking-water supplies. In addition the survey is expected to shed some light on what level of treatment may be needed to achieve a desired risk level in the finished water
When Method 1622 has been finalized, EPA says it will not abandon looking for alternative and complementary methods for detecting Cryptosporidium. According to Regli, "Method 1622 will be considered in the context of other methods that can be developed in the next few years." The ICR survey results will be considered along with alternative methods to ultimately shape the longer-term Enhanced Surface Water Treatment Rule (ESWTR), expected to be finalized in 2002.
One approach being considered for regulation is to base the level of treatment required on total oocyst counts. Many believe that this approach—reporting all detected oocysts—is too conservative, resulting in unnecessary treatment at great costs to the water-purification plants. According to Stig Regli of EPA's Office of Ground Water and Drinking Water, cost savings could be enormous if viability and speciation of the detected oocysts were considered in lieu of total counts, because not all detected oocysts are infectious to humans. By identifying how many of the detected oocysts are potentially harmful some utilities dependins? on their watershed characteristics could experience as much as a 1- to 2-log (ten- to 100-fold) reduction in the oocvsts they might be reqtiired to treat remarks Regli
Tied in with the ICR is another piece of legislation intended to reduce the levels of disinfection byproducts in drinking water, which could lead to lower amounts of chemical treatments. "Given that there could be a very substantial driving force toward lowering byproducts, there's that greater need to have tighter control for the pathogens," warns Regli. EPA held a workshop last October to learn what types of methods are currently being developed by leading researchers in the field, giving the experts an opportunity to present their cutting-edge research. "The methods presented at the meeting all have potential to be applied in some combination of a method or several methods for the longer-term ESWTR depending upon the regulatory option that ultimately is selected," says Regli.
50 A
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Analytical Chemistry News & Features, January 1, 1998
Percoll-sucrose cushion The oocysts are then collected on a cellulose-acetate membrane (CAM") and stained with fluorescent labeled monoclonal antib di F l1 the i v. i
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scope, equipped with epifluorescence ulumination and differential interference contrast or Hoffman modulation optics. Given the number of steps involved in the IFA procedure, it is no wonder that poor recovery efficiencies have been reported. '"The greater the number of steps in a method, the lower the efficiency. We are trying to minimize the number of steps," says Colin Flicker of Thames Water Utilities (U.K.). Analysts familiar with the method, however, do not agree on which step is responsible for the low efficiencies. "The greatest loss occurs during centrifugation," says Ricardo DeLeon of the Metropolitan Water District of Southern California. Other lysts say that the flotation step is to blame Thaddeus K Graczyk and co-workers of The Johns Hopkins University School of Hygiene and Public Health believe that the greatest loss of oocvsts occurs during processing of the filter They are investigating a CAM-filter dissolution method that involves the rnmplete di^nlutinn of the membrane
for better recovery of the oocysts The CAMfilter dissolution recovery proced reported to have no effect on the viability or infectivity of Cryptosporidium parvum oocysts, the infectious form of Cryptosporidium.
Molecular probes have recently hit the scene, and many microbiologists are excited about their ability to distinguish between the different species of Cryptosporidium. "Perhaps the greatest strength of fluorescent in situ hybridization [FISH] probes is their ability to rapidly differentiate to the species level," says Alan Iindquist of EPA's Office of Research and Development. "The commercially available antibodies are not speciesspecific. Other molecular techniques, such as polymerase chain reaction [PCR], require more time and are faced with inhibition problems from components in the water." Iindquist is evaluating molecular probes for identification of protozoan parasites and for species confirmation A FISH probe is one example of a molecular probe being used to studv Crvbtosboridium oocvsts and Giardia cysts FISH probes can be surrogate indicators of viability, provided that the appropriate probe target is selected. According to Iindquist, ribosomes are a common target for FISH probes for several reasons: They are relatively conserved within a species, they are stable, they are essential for the production of proteins, and they are present in multiple copies. The FISH probe fluorescently labels ribosomal RNA which can then be visualized under a fluorescent microscope. Ribosomal RNA is belleved to degrade rapidly in oocysts that are nonviable; thus only viable oocysts wiil be ffuorescenuy labeled. "I don't know that I believe that FISH resembles viability because I know how stable those ribosomes are especially inside the oocyst Until we know what the degradation rate of ribosomes is I don't know whether we can say anything viability " comments water pollution microbiologist loan Rose of the I Iniversitv of South Florid S me disa "There l tsof data th fi di ate th t 'h somal RNA ro•J
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vides a good indicator of viability for oocysts from the environment. What is questionable is if rRNA will be useful for measuring viability after disinfection, says one expert. Another method for assessing viability is in vitro cell culture. The procedure involves subjecting Cryptosporidium oocysts to bleach treatment that destroys other living organisms, leaving behind viable oocysts, which can then be directly introduced into a tissue cell line without con-
tamination. "Cell culture is not just being used in our lab; it's been used in five or six other labs now. Each of us used different cell lines and slightly different methods for detection of the infection process, and we all found similar results. That tells me this is a robust method," says Rose. "The bottom line is that cell culture works. With young oocysts, we can get detection of infection with one oocyst." Rose agrees with many other researchers who believe that viability need not be tested on a routine basis. "I think there are occasions when we need to test viability." She envisions having four or five labs in the United States that can test viability when it is necessary. The key question is, when is viability testing going to be necessary? "We need to define that," emphasizes Rose. "Cell culture is not something that a routine water lab can do." Rose does believe
'We're getting to the point where it's not just research anymore. You can actually buy a kit." that cell culture has a place in specialized studies and that it will start being used in place of animal infectivity studies, in disinfection work, for example. Some organisms have distinct biochemical markers, much like fingerprints, that can aid in their identification. David White of the Center for Environmental Biotechnology, University of Tennessee, and co-workers at Microbial Insights have found a signature lipid biomarker that is unique to Cryptosporidium oocysts, which may alao correlate with infectiousness. The method for determining its molecular structure (10 hydroxy stearic free fatty acid) involves collecting the oocysts and extracting the lipids with an organic solvent. The free fatty acid fraction is then separated by HPLC and detected as negative ions by MS following electrospray ionization The procedure has the sensitivity to detect less than a single oocyst even from
a dirty environmental sample, says White. "The real challenge is to get that signal out from all the other signals in an environmental sample at low levels of detection," says Rose. "I think we have underesttmated that type of instrumentation. I'm looking at UVvis spectroscopy right now, for identification of microbes. I see instrumentation such as MS as having great potential, but it is in the hypothesis testing stage. I see it 10 years down the road." Several labs are currently evaluating vortex flow filtration (VFF) and immunomagnetic separation (IMS) as methods for concentrating and separating protozoan parasites. Joe Crabb of ImmuCell Corporation and collaborators of Thames Water Utilities (U.K.) describe the principle behind VFF, 'Water is passed through a cylindrical membrane filter, which is rotating at high speed within an outer chamber. The rotation of the membrane sets up waves of turbulence in the sample passing through the chamber known as Taylor vortices These continuously 'scrub' the surface of the membrane keeping it free from particulate matter and thus preventinp1 blockaffes while increasing the volume of sample that can be concentrated " advantage to VFF can set water sample to be concentrated and walk away "I think the instrument has the potential to be on line where you are contin ally concentrating, but it needs some engineenng in that regard, says Rose. ImmuCell claims that its system can be set up to concentrate 10-100 L at a time. ImmuCell is involved in a joint venture with New Jersey-based Membrex, to develop a Crypto-Scan water diagnostic test. According to a press release issued in June, "The Crypto-Scan test utilizes VFF technology to concentrate water samples, followed by an IMS technology to further concentrate and clean up the samples for detection of Cryptosporidium oocysts." Crypto-Scan is one of the techniques being evaluated for Method 1622. IMS is not a new method. "Frank Schaeffer of the EPA was die first to publish its use for Giardia, ,nd we eublished on its use with Cryptosporidium ana detection with PCR about four years ago," says Rose. "It has taken a while to get to the point where we can get a kit and reagents
Analytical Chemistry News & Features, January 1, 1998 51 A
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and a magnet. Many variables have to be worked out with the magnetic-separation technique—the strength of the magnet, the size of the beads, whether the beads are coated, the type of antibody, and the way the antibody is attached to the beads, emphasizes Rose. "I'm glad to see that companies are taking an interest in developing a standardized approach a way to look at QA/QC. It's not perfect ye,, but we're getting to the point where it's not just research You can actually buy a kit." Rose believes that no matter what collection and detection methods are used, IMS in the middle of the procedure will improve the method overall. "In my comparisons and in others', IMS has equaled flow cytometry. It is low cost. You can do it on the bench—anybody can do it. As a technique for monitoring contamination in the environment, whether it be food or water, it is a technique that has great potential. I think it will be the cornerstone." Many are optimistic about continuousflow cytometry with fluorescent cell sorting as a method for separating and purifying Cryptosporidium oocystss ThT Macquarie University Centre for Analytical Biotechnology (MUCAB) (Australia) and the Australian Environmental Flow Cytometry Group have been developing flow cytometric methods for detecting Cryptosporidium for about five years. The primary disadvantages of flow cytometry are the high cost ($100,000-$200 000) and the limited availability of the instrument in the United States. Most water utilities in the United Kingdom own a flow cytometer specifically for water analysis. The instrumentation is reliable and has been in use routinely since 1990 in the United Kingdom and Australia, says Duncan Veal of MUCAB. "The existing instrumentation is expensive and difficult to use but has proved cost-effective for processing a large number of samples." The Australian group has developed a simple-to-use (and quick) reagent kit for Giardia and Cryptosporidium that will run un simple-to-use, off-the-shelf instruments, designed for routine clinical analyses. The kits provide the option of determining viability and speciation Jon Standridge of the University of Wisconsin, State Laboratory of Hygiene, sur-
5 2 A Analytical Chemistry News & Features, January 1, 1998
veyed 18 medical facilities with flow cytometers—primarily used as an AIDS diagnostic tool—to determine their willingness to run samples for the water utilities. Fifty percent said they would be willing to offer their services. Does the potential exist for cooperation between the medical facilities and the water treatment plants? Some experts are skeptical. "The hospitals do not want to take your environmental samples. You have to have a dedicated instrument for specific uses," says Rose. Standridge disagrees, "The instrument we do our research on is routinely used for clinical work as well." Mark A Borchardt and Susan K. Spencer of Marshfield Medical Research Foundation (Wisconsin) evaluated oocyst recovery efficiencies with a blood-cell separator—a device used in the medical industry to fraction and collect blood components. The apparatus gave high recovery efficiencies and has the potential for continuous sampling. It also has the added advantage of being nonspecific to Cryptosporidium. The device will likely be effective with other protozoan pathogens, says Borchardt. The main drawback of the bloodcell separator for routine water testing is its low availability and high cost ($40 000$50 000) However a Milwaukee-based company Molecular Biology Resources has developed a prototvrje with a much lower price tao' (estimated on the order of thousands of dollars) than the blood-cell eliminating m a n y r»f ttif> crip
cial features that were designed with patient safety in mind In addition to solving the analytical problems associated with Cryptosporidium detection, other issues must be considered, such as where to sample, how often to sample, the volume to sample, and what precautions must be taken during extreme events. "I think a lot of storm waters and wastewaters impact our drinking water supplies," says Rose. "No matter how good we are with treatment, if extreme storm events are associated with our outbreaks, we've got to recognize that and learn how to deal with extreme events," she warns. "This year could be interesting, because El Nino is causing floods and more droughts the exact conditions that have been associated with waterborne outbreaks " Britt Erickson
