FEATURE
New Cryptosporidium
Testing W Methods Better methods for detecting waterborne microbes in drinking water advance as EPA begins nationwide data collection.
MYRNA
E.
WATANABE
5 3 2 A • VOL. 30, NO. 12, 1996 / ENVIRONMENTAL SCIENCE & TECHNOLOGY / NEWS
h e n EPA p u b l i s h e d in 1994 its intention to collect information on the prevalence of the protozoans Giardia a n d Cryptosporidium in drinking water by monitoring public water supplies (2), water microbiologists were u n h a p p y with t h e p r o posed analytical m e t h o d for Cryptosporidium. The collection procedure and immunofluorescence assay required in EPA's information collection rule (ICR) h a d m a n y deficiencies. " I t ' s v e r y e x p e r i e n c e - i n t e n s i v e , it's t i m e consuming, it's expensive, it doesn't give you information on viability and infectivity of organisms, and it doesn't tell you the species you're detecting," asserted microbiologist Walter Jakubowski of EPA's National Exposure Research Laboratory in Cincinnati. And in dealing with a waterborne pathogen like Cryptosporidium parvum, a n analytical test that does not tell whether the organism is present could be a serious public health threat: in its worst outbreak in Milwaukee in 1993, the microbe infected 403,000 people and contributed to 100 deaths (2). Ingested Cryptosporidium causes gastrointestinal s y m p t o m s a n d illness t h a t c a n b e c o m e lifedireatening in individuals with impaired i m m u n e systems, such as cancer and AIDS patients. Ingestion of only one Cryptosporidium oocyst, the infective life stage of the organism that contains four sporozoites that begin to reproduce in the living host, may lead to infection. Even EPA, in its final promulgation of the ICR in May this year (3), acknowledged that the m e t h o d it is requiring is faulty and needs replacement or refinement. Researchers worldwide are addressing this need, some with funding from the American Water Works Association Research Foundation in Denver, the U.S. EPA, a n d the D e p a r t m e n t of Agriculture. 0013-936X/96/0930-532A$12.00/0 © 1996 American Chemical Society
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Many of these developing methods rely on sophisticated techniques to identify organisms through isolation of their nucleic acids, though a few new methods are fairly simple changes to die protocol and may improve something as basic as concentration of the water sample (4). Although too late to be included in the collection rule, other methods to filter, concentrate, and detect Cryptosporidium may be ready for use by the time EPA completes collection of data on the prevalence of the microorganism and is ready to regulate monitoring and testing. Scientists and regulators have little information on the distribution and occurrence of Cryptosporidium, Giardia, and viruses in U.S. waters. The collection rule requires water utilities that treat surface water and serve more than 100,000 people to monitor influent water for 18 months (3). A utility that identifies 10 or more Cryptosporidium oocysts per liter of water during the first 12 months of monitoring must also monitor treated water. The immunofluorescence assay method for Cryptosporidium stipulated in the ICR is similar to one that had been proposed by the American Society for Testing and Materials at the time EPA was selecting a method (5). This technique uses monoclonal antibodies raised against a protein present in Cryptosporidium oocysts. When introduced to a sample containing Cryptosporidium oocysts, the antibody attaches to the organism. This sample then is treated with a secondary antibody that itself is attached to a molecule that will fluoresce. This secondary antibody will attach to the now-joined monoclonalCryptosporidium oocyst, making a three-part sandwich. When examined under epifluorescence microscopy, using an ultraviolet light source the sandwich fluoresces.
ICR method questioned Because so many researchers questioned the validity of this method, EPA sponsored several filter seeding tests of water samples with Cryptosporidium oocysts to determine the technique's efficacy. The test results were disappointing, yielding a mean percent of oocyst recovery of 23% in the first tests and 35% in the second tests, with a range of 0-138% recovery. There also were some false positives, where an organism mat was not Cryptosporidium was identified as Cryptosporidium, and false negatives, in which Cryptosporidium present in the sample was not identified (6). These results almost derailed the plan to include the immunofluorescence assay for detecting Cryptosporidium in the ICR (7) and made it imperative that for long-term monitoring and for testing of water supplies, more reliable methods be found. The immunofluorescence assay is just one step of a multipart ICR-designated method, and other steps of the method have drawn complaints. To test for the presence of Cryptosporidium in reservoiis and surface waters, large volumes of water first must be concentrated, generally on a filter, according to Ellen Braun-Howland of the Wadsworth Laboratories, New York State Department of Health, Albany. For source water, 100 L is taken for sampling; more than 1000 L may be sampled for treated water (8). Filtering and concentrating the Welter samples are time-consuming, and Braun-Howland pointed out that the greater the size of the sample, the more Cryptosporidium oocysts are likely to be lost in the process. Senior microbiologist Steve Schaub of the EPA Office of Water's Office of Science and Technology explained that "gunk" collects on the filters, and only about 1 mL of this material is taken from the filters for assay. But if only 1 mL is assayed and 49 mL remain untested, an estimation of the number of oocysts within the filtered sample may be very inaccurate. The second step is washing the filter and concentrating and purifying the oocysts, usually in a buoyant density gradient. Again, Braun-Howland explained, "Cryptosporidium sticks to everything. It sticks to particulates. You take a big loss on that step." The third part is the immunofluorescence assay to identify the Cryptosporidium oocysts. After being treated with the fluorescent antibody, the oocysts must be identified and counted by a highly trained microscopist. Victor Tsang, chief of the immunology branch of the Division of Parasite Diseases, Center for Infectious Diseases, Centers for Disease Control and Prevention in Atlanta, pointed out that the whole ICR procedure is extremely timeconsuming, with the microscopy step taking many hours. He also explained that the test may require significant handling of samples by personnel, exposing them to potentially infectious material in initial sampling stages. Even when the oocyst is identified microscopically, there is no way to tell whether it is viable or already dead. This differentiation is important when monitoring fully processed water, because it tells the facility how successful its water treatment method is in destroying Cryptosporidium. Furthermore, as Ricardo DeLeon of the Metropolitan Water District VOL. 30, NO. 12, 1996/ENVIRONMENTAL SCIENCE & TECHNOLOGY / NEWS • S 3 3 A
Finding Crypto: dead or alive?
The process of identifying Cryptosporidium oocysts in drinking water samples is only part of the detection challenge; determining whether they are alive and therefore a health threat is a tougher problem. The EPA-approved immunofluorescence assay currently in use identifies the outside wall of the oocyst (A) but provides no indication whether the sporozoites inside are viable. Staining the same organism with different fluorescent molecules provides a look inside. The DAPI stain (B) highlights DNA (two bright points), and a rhodamine tag (C) labels rRNA throughout the sporozoites, both indicators that the sporozoites may be viable. These new labeling techniques are being evaluated by researchers at the New York State Department of Health. (Photos courtesy Ellen Braun-Howland, New York State Department of Health)
of Southern California points out, even skilled microscopists may misidentify a microorganism.
New detection methods New methods, however, are on the horizon. Jennifer Clancy of Clancy Environmental Consultants, St. Albans, Vt, who performed the immunofluorescence assay validation tests, observed that some detection methods are further along than others. One such technology, flow cytometry, has been in use in the United Kingdom for years but is still under development in the United States. In flow cytometry, cells are stained with fluorescent dye attached to a monoclonal antibody as in the immunofluorescence method. But then the sample is analyzed not by a microscopist, but by a cell sorter that checks for cells of a certain size, fluorescence, and internal structure (8). Those cells that the sorter picks out must then be examined by microscopy. Problems with flow cytometry are cited by the Centers for Disease Control's Working Group on Waterborne Cryptosporidiosis, a group of researchers in the field formed as an outgrowth of a workshop held in September 1994 and headed by EPA's Jakubowski (8). These problems include losses related to sampling technique, which are not addressed by new detection technologies; personnel requirements for running the flow cytometer; difficulty in obtaining the equipment; and the continued need for confirmation of oocyst identification by microscnpy. Colin Fricker of the U.K.'s Thames Water Utilities Ltd. in Reading has been working with flow cytometry systems for six years. He is now developing a vortex flow filtration method that will contain an absolute filter that will trap particles >500 kilodaltons, as opposed to the nominal 1-u filters currently in use. Fricker said that there is "no possibility for Cryptosporidium [which is 4-5 u in diameter] to pass through that filter." One of the major problems with all the antibodybased methods is that the monoclonal antibodies in use are not specific enough and bind to a glycosylated portion of protein, Fricker explained. Because they bind to a portion of a molecule containing a car5 3 4 A • VOL. 30, NO. 12, 1996 / ENVIRONMENTAL SCIENCE & TECHNOLOGY / NEWS
bohydrate that may be found in cells other than Cryptosporidium, these antibodies also cross-react with algae and many other organisms. Fricker is working with ImmuCell in Portland, Me., to raise a polyclonal antibody that will specifically attach to a region on a protein on the oocyst wall. This polyclonal antibody will be specific for the oocyst protein and will allow separation of Cryptosporidium oocysts srom the rest of the filtrate. Identification will be done using a monoclonal antibody. Refinements of the immunofluorescence assay technique that are within six months of reaching the market, according to Clancy, include immunomagnetic separation (IMS) methods. IMS methods will allow the separation of Cryptosporidium from other organisms. Antibodies to the Cryptosporidium oocyst are tagged with iron and introduced into the sample, and then the sample is run through a magnetic field. This should separate the tagged cells, presumably oocysts, from other organisms and debris. Fricker, Clancy, and colleagues now are working with IGEN, Inc., of Gaithersburg, Md., on refining an IMS system that Fricker believes will have the capacity to be used in the range of detection of 3. single oocyst. Risot now he explained, the detection limit is 30 oocysts in a sample which is not sufficient because even one viable oocvst may cause infection in a human "The ICEN assav is a screening test" said Fricker "that rapitilv screens larpp numhprs of samples to
