analyzing for cryptosporidium - ACS Publications - American Chemical


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ANALYZING FOR EPA scrambles to refine an analytical method CRYPTOSPORIDIUM

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ince 1993 the U.S. Environmental Protection Agency (EPA) has been under intense pressure to regulate the waterborne pathogen Cryptosporidium in drinking water. That was the year that Cryptosporidium slipped through Milwaukee's water purification system, sickened an estimated 400,000 people, and contributed to 104 deaths. Yet regulations on Cryptosporidium probably will not be finalized until late 1997 at the earliest, says Stig Regli of EPA's Office of Ground Water and Drinking Water. And, admits Regli, the delay is largely the result of problems with EPA's analytical procedure to detect Cryptosporidium and another troublesome drinking-water pathogen, Giardia lamblia. The delay has important public health

As concern increases, no method satisfactorily detects the waterborne pathogen implications. There is no medical cure for Cryptosporidium infection. Those stricken develop flulike symptoms and diarrhea that can last for one to two weeks. Moreover, the very young and the elderly along with immunocompromised or immunosuppressed individuals can develop life-

threatening cholera-like symptoms that can linger for months. In June, EPA and the Centers for Disease Control issued drinking-water guidelines for people with weakened immune systems that recommend regularly boiling water or fitting water taps with filters. At the same time, concerns over Cryptosporidium are being played out in the political arena as Congress and the Clinton administration spar over new versions of the Clean Water Act and the Safe Drinking Water Act. Members of the administration, including the president, regularly invoke the Milwaukee Cryptosporidium outbreak in speeches attacking what they see as congressional efforts to weaken these environmental laws. Others cite EPA's failure to regulate Cryptosporidium

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Focus as evidence that the agency has squandered its resources on developing regulations and analytical methods for what they say are less risky chemical contaminants. Since 1984 EPA has documented several Cryptosporidium outbreaks in the United States, including a 1994 event linked to the deaths of 35 HIV-infected individuals in Las Vegas, NV. Each outbreak has occurred at a utility that complied with EPA's drinking-water regulations. European waters are also infected with the pathogen; more than 300 people were stricken this August in Devon, U.K. Preliminary studies suggest that Cryptosporidium is already widespread in U.S. surface waters. EPA has estimated that more than 155 million U.S. citizens are at risk for infection from their drinking water. Yet the scale of the problem is unknown because of the lack of analytical data. "Only a few water quality and public health laboratories have the capability to evaluate any microbial contaminant, and most can perform only the simplest test for fecal coliform," says microbiologist Joan Rose of the University of South Florida. Problems and disappointments Cryptosporidium survives in environmental waters as ~ 5-pm sized oocysts—spherical or egg-shaped particles surrounded by a tough protective coat. Ingestion of the oocysts starts a complex life cycle that leads to infection of the small intestine by the protozoa and eventually the excretion of a new generation of oocysts. Unfortunately, standard drinking-water disinfection practices such as chlorination are ineffective against Cryptosporidium oocysts. Instead, water utilities reduce the turbidity of their treated water by flocculation, sedimentation, and filtration as a means of eliminating the micron-sized Cryptosporidium. Last year the American Water Works Association urged its members to lower the turbidity values in their finished drinking water from EPA's regulated value of 0.5 NTU (nephelometric turbidity units) to 0.1 NTU. EPA had initially planned to issue similar values as a short-term regulation this year, but the Republican congressional victories and the backlash against federal controls halted that approach. Instead, EPA has launched a cooperative, voluntary safe drinking-

water initiative to aid utilities in lowering their turbidity values. Meanwhile, EPA has had to work hard to solve problems with its analytical method for protozoa. EPA's method (which is based in turn on an American Society for Testing and Materials [ASTM] method) collects Cryptosporidium oocysts and Giardia cysts by passing large volumes of water, as much as 100 L of raw water or 1000 L of finished water, through yarn-wound polypropylene cartridge filters. Retained particles are eluted, concentrated by centrifugation, and further separated from unwanted debris by flotation on a Percoll-sucrose cushion. The concentrated organisms are collected on a cellulose-acetate membrane and stained with monoclonal antibodies tagged with fluorescein. The labeled pathogens are viewed

EPA specified everything in the methodfrom brand-name reagents to when slides should be cleaned. under a microscope equipped for epifluorescence and differential interference contrast or Hoffman modulation optics. A trained analyst classifies the cysts and oocysts according to size, shape, and internal morphologies. These characteristics must be identified under the microscope in green fluorescing pathogens scattered among interferences such as algae, which also fluoresce, or turbidity. Even at its best, the method known as indirect fluorescent antibody (IFA) fails to determine whether the oocysts or cysts are alive or not (viable or nonviable) or differentiate among different species of Cryptosporidium. (Not all species lead to illness in humans.) Tests sponsored by ASTM with commercial laboratories in 1991 and 1992 found that the procedure leads to widely varying results. A second study run by the Erie County Water Authority and re-

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ported in 1994 revealed that 6 of the 12 participating laboratories failed to detect any oocysts, and the rest of the labs recovered an average of 2.8% of the oocysts from the spiked samples (I). These results led to the development of a more detailed procedure, specifying everything from brand names of reagents to when slides should be cleaned with a Kimwipe. In addition, workshops were instituted to train analysts to recognize oocysts. EPA also plans to institute a laboratory approval program. However, an EPA-run round-robin performance evaluation this January of the revised method involving nine "expert" commercial laboratories yielded recoveries that ranged from 0 to 130%. These results fell short of EPA's average recovery goal of 20% for Cryptosporidium in spiked samples of environmental waters. Results for Giardia proved better, with a "passing" grade of more than 50% average recovery, although precision was also poor. Recent analyses of the overall analytical procedure differ as to where most of the oocysts are lost; one report cites the clarification and centrifugation steps (2), whereas another study blames the flotation step (3). The basic message, says research microbiologist Frank Schaefer of EPA's Office of Research and Development in Cincinnati, OH, is that the more you manipulate the sample, the more likely you'll lose analyte. In response to the disappointing Cryptosporidium results, EPA microbiologists along with experts from academia and industry held a series of meetings and workshops this year, including a major meeting on May 18 to "refine" the method. According to Schaefer, participants at the meeting agreed to remove the remaining options in the IFA procedure to reduce variations among laboratories. For example, analysts will no longer have a choice on how to prepare the Percoll-sucrose cushion. Participants also agreed to test some comparisons on small variations in the method such as using well slides instead of the cellulose membrane in the microscopic analysis. With the changes in place, a second round-robin study using 12 laboratories began in September, and additional tests requiring field collection and spiking of water

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< < Can you spot the microbes? Left: a photomicrograph of a green fluorescing Giardia cyst. Right: Giardia and Cryptosporidium scattered among typical raw water debris.

samples from 7 sites were launched in October. "We are hoping that the [average] recoveries will go up 10%," says senior EPA microbiologist Stephen Schaub, "and that we will be able to determine values within [a factor of 10] at any particular site." Regulations on hold Most of the details of EPA's protozoa method are spelled out in a regulation the agency formally proposed in February 1994, known as an information collection rule (ICR) (4). The ICR would provide EPA with the data it needs to assess the scale of the pathogen problem. It accomplishes this task by requiring all public drinking-water utilities that draw water from sources likely to contain the pathogens (i.e., surface waters or groundwaters that are closely tied to surface waters) and serve 10,000 or more customers to test their raw waters for the pathogens. Large utilities that supply more than 100,000 people would also need to test their finished drinking water. The ICR would also require monitoring for disinfection byproducts, such as trihalomethanes, which are formed during the water treatment process, and total culturable viruses. It also dictates the collection of relevant engineering data. Completion of the ICR-mandated monitoring, in turn, will help shape EPA's final version of the enhanced surface water treatment (ESWT) rule, the long-awaited regulation covering Cryptosporidium, as well as providing new restrictions on Giardia (5). In addition, the ICR is tied to another proposed rule putting new limits on disinfection byproducts (6). ESWT and the disinfection byproduct rules were proposed in July 1994. At that

time EPA had expected to begin monitoring under the ICR in October of that year and complete the work by March 1996. Instead, the ICR will probably be promulgated this month, says Regli, regulation manager for the ICR. That will allow monitoring to begin in March or April, just in time for the 1996 spring runoff. Regli is confident that the ICR will include Giardia analysis, but whether the analytical method will be sufficiently modified in time to include Cryptosporidium is questionable. EPA watchers say that whether the analytical method is complete or not, Robert Perciasepe, head of the Office of Water, will not allow any further delays in the ICR. On the other hand, warns Dan Pedersen of the American Water Works Association, "If the method [in the final ICR] is as lousy as it's been, we will be involved in litigation." Regli says that if the method is not approved for Cryptosporidium in the final ICR, EPA is considering working with experienced microbiology research laboratories, like Rose's, to survey statistically chosen sites for the pathogen and use the results as a national model. Alternative methods If the IFA method is so problematic, why is EPA pursuing it? Because it is the best method available, points out Rose. "It is used in many laboratories and has been published in over 50 articles," she adds. "There is a good scientific base." EPA says it does not need significant accuracy in its protozoan analyses because the treatment strategies that water utilities use are triggered by tenfold changes in concentration. For example, EPA regulations now require utilities to re-

move or inactivate at least 99.9% (3-log) of the Giardia cysts in raw source waters. Regulations concentrate on the treatment side because it is not possible to routinely monitor pathogens in rapidly flowing finished drinking water. The concern over Cryptosporidium and the limitations of IFA, however, have sparked research on alternative methods. Any new method will require high sensitivity because Cryptosporidium and other protozoan parasites lead to infection even at a low dose. Recently, a value of > 10-30 oocysts/100 L of finished water was proposed as the level at which utilities would issue an alert (7). In Rose's and several other laboratories, the polymerase chain reaction (PCR) is used to confirm Cryptosporidium detection, but Schaefer says that EPA has found problems with routine PCR: "In pure water it gives excellent results, detecting as few as one Giardia cyst. But in natural waters, inhibitors like humic and fulvic acid chelate the magnesium needed for the Taq PCR (8)." He also worries about amplifying unwanted DNA in the environmental samples. Rose's group is also using antibodycoated magnetic beads to clean up samples before PCR amplification. She predicts that these magnetic bead antibodies will eventually be incorporated into the IFA method as a better cleanup procedure. However, EPA has discovered that turbid solutions and environmental waters containing iron compounds inhibited the affinity of these magnetic particle antibodies. EPA is still investigating PCR as a method, says Schaefer, but the agency has abandoned magnetic antibody research because to make it work required "excessive manipulations" (9). An enzyme-linked immunosorbent assay (ELISA) is probably the quickest test for pathogens and is already used in clinical applications. Recent work suggests that an ELISA can be used to identify as few as 1 to 10 targets in environmental samples, but, warns Schaefer, it only detects and does not confirm. Schaefer is more enthusiastic about the application of flow cytometry with fluorescent cell sorting which, in principle, detects a single oocyst or cyst. Although the instrumentation is expensive, this sophisticated technique is routinely used at

Analytical Chemistry, December 1, 1995 733 A

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Focus several water utilities in the United Kingdom with reported overall recoveries of ~ 30% for Cryptosporidium (10). Microbiologists worry that many of the oocysts detected in the above methods are not viable and that the numbers are therefore misleading. One method in development that does determine viability is based on a technique called FISH (fluorescent in situ hybridization), which labels a section of ribosomal RNA with a complementary RNA fluorescent probe. Ribosomal RNA degrades quickly in nonviable cysts or oocysts, leaving FISH RNA probes to mark primarily viable cells. FISH can be combined with confocal microscopy to look at samples simultaneously marked with multiple fluorochromes. For example, says Schaefer, it is possible to fluorescently label the surface of Giardia cysts and the ribosomal RNA. Identification and confirmation are thus combined in one step. Alteratively, FISH can be used with flow cytometry to select viable protozoa. Microscopy combined with imaging by a cooled, slow-scan charged coupled device provides another way to look at pathogens tagged with multiple fluorescing probes. This technique works with lower magnification microscopy, but the big limitation so far is the software, says Schaefer. In vitro cell culture is another route for detecting viable infectious forms of Cryptosporidium. Following a purification process such as that used in IFA, the sample would be exposed to chlorine to kill off everything but the tough oocysts and then prompted to begin its parasitic life cycle by introduction into a tissue cell line. Initial work indicates the method requires as few as 100 oocysts for a response. Other methods to test viability rely on the simultaneous exclusion and inclusion of certain dyes, or an enzyme test. Some scientists express high hopes for a method called electrorotation assay in which oocysts bind to the antibody-coated particles, which are then spun in a rotating ac electric field. Bound and free particles spin at different rates. The method reportedly also differentiates between viable and nonviable oocysts. Most research microbiologists are skeptical of these claims, however, pointing out that the procedure has not been published in the peer-reviewed literature or exhaustively

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tested. "It's an entirely new method and each step will need to be checked," says Schaefer. Nevertheless, Hach Chemical (Loveland, CO) plans to offer a reagent kit for the method early next year, according to company microbiologist Jill Plyter. "We feel very confident about the method," says Plyter. And, she confirms, it will discriminate among viable and nonviable pathogens. None of these analytical methods replaces turbidity as a real-time measure for pathogens. At best, the direct detection methods require 24 hours—too long for water utilities, which must keep treated water flowing. However, turbidity measurements do not protect against protozoa in lowturbidity waters. Although still an indirect indicator of pathogens, particle counting is currently under investigation as a better means of real-time analysis because the technique determines particle sizes and thus warns of particles in the size range of oocysts and cysts. Rose warns that other analytical issues need to be resolved, such as where to sample, how sensitive a method must be to provide meaningful data, and how often to sample. "I can measure log reductions [in Cryptosporidium concentration]," she says. "But are they real reductions?" Alan Newman References (1) Clancy, J. L.; Gollnitz, W. D.; Tabib, Z. J.Am. Water Works Assn. 1994,86(5), 89-97. (2) LeChevallier, M. W. et al. Appl. Environ. Microbiol. 1995, 61, 690-97. (3) Nieminski, E. C; Schaefer, F. W.; Ongerth, J. E. Appl. Environ. Microbiol. 1995,62,1714-19. (4) Fed. Regist. 1994, 59, 6332. (5) Fed. Regist. 1994, 59, 38832. (6) Fed. Regist. 1994,59, 38868. (7) Haas, C. N.; Rose, J. B.J. Am. Water Works Assn. 1995,87(9), 81-84. (8) Rodgers, M. R.; Bernardino, C. M.; Jakublowski, W. Water Sci. Technol. 1993,27,85-88. (9) Bifulco, J. M.; Schaefer, F. W. Appl. Environ. Microbiol. 1993,59, 772-76. (10) Watkins, J.; Kemp, P.; Shepard, K. In Protozoan Parasites and Water; Betts, W. B. et al., Eds.; Royal Society of Chemistry: Oxford, U.K., 1995; pp. 115-21. Suggested reading Protozoan Parasites and Water; Betts, W. B. et al., Eds.; Royal Society of Chemistry: Oxford, U.K., 1995.