By Randi M. McCuin

Summary: Two more familiar, not to mention dangerous, waterborne parasites are Cryptosporidium and Giardia. Much focus has been directed at reducing their presence in various water sources. Formulating the right method involves many evaluations and tests, which may take years of research and monitoring. A few steps, though, have been taken to that end.

Giardia and Cryptosporidium are protozoan parasites found in surface waters worldwide. These organisms have been implicated in numerous waterborne disease outbreaks. Outbreaks have occurred following consumption of unfiltered water, with or without disinfection, as well as with disinfected, filtered water supplies. Symptoms of giardiasis and cryptosporidiosis are similar and include nausea, profuse, watery diarrhea and a low-grade fever. Therapeutic drugs are available for persons infected with giardiasis, but there are no known remedies for cryptosporidiosis. This disease is self-limiting in healthy individuals but can lead to death in immunosuppressed individuals.

Since the early 1980s, drinking water suppliers and the scientific community have known chlorine could inactivate Giardia cysts at the appropriate concentration, pH and contact time. Waterborne outbreaks attributed to the parasite since have largely been limited to untreated drinking water sources and from sources other than water. While chlorine is effective for inactivation of Giardia, Cryptosporidium oocysts are very resistant to chlorine disinfection. Currently, the only effective treatments to inactivate oocysts are UV, ozone and chlorine dioxide.

Costs of methods
Crypto and Giardia are present in many wild and domestic animals and, therefore, agricultural runoff and wastewater are thought to be significant sources for surface water contamination. Analytical methods to determine concentration of these organisms in these matrices are costly, labor intensive and require technical expertise. The Information Collection Rule (ICR) method involves the filtration of several liters (L) of water through a 1-micron (µm) nominal porosity filter. The retained particles are eluted from the filter material with phosphate buffered saline-containing detergents. The filter wash is concentrated using centrifugation and the pellet material is subjected to a non-specific density gradient flotation. The floated material is stained with fluorescein isothiocyanate-labeled antibodies (FITC-mAb) specific to Crypto and Giardia.

Problems associated with this method are filters are inefficient at capturing and retaining Cryptosporidium oocysts; the separation process is non-specific, allowing for separation of many objects that cross-reacted with the FITC-mAb, and only a small portion of the sample can be examined microscopically for the presence of these organisms. Most laboratories have abandoned this method with development of a new validated method that’s less labor intensive and provides more accurate and consistent results.

Effects of the method
In 1999, the U.S. Environmental Protection Agency approved “Method 1623: Cryptosporidium and Giardia in Water by Filtration/IMS/FA,” which was developed with commercially available technologies. The main objectives of this method were to collect a small sample volume (10 L) with a more efficient filter, selectively isolate the oocyst and cysts from the contaminating debris and examine microscopically the entire volume collected. The method was validated under Performance Based Measurements System (PBMS) guidelines set by the USEPA. Simply stated, PBMS is a mechanism for laboratories to validate a method alteration providing that the quality assurance/quality control (QA/QC) guidelines established in the original method are met or exceeded. PBMS has a two-tier validation scheme. Tier 1 validation is carried out by a single laboratory. Changes can be made in components (filter, IMS kit, mAb, etc.) or protocols. As long as the overall method recovery meets or exceeds the established QA/QC, those modifications to the method are approved for use in that laboratory. A Tier 2 validation involves the modification study being conducted in three laboratories.

Results from at least 75 percent of the participating laboratories must meet the established limits and, if acceptable, the method modifications are approved for use by all laboratories. Method 1623 was validated by the USEPA in a 12 laboratory round-robin study using the Envirochek capsule filter (Pall Corp.), and Dynabeads GC-Combo immunomagnetic separation (IMS) kit (Dynal A.S.). Since that time, additional filters have been validated for use, including the Whatman CrypTest and IDEXX FiltaMax. A second FITC-mAb, Waterborne Inc.’s Aqua-Glo, has also been validated. As other commercial products enter the market, the PBMS system is a cost effective way for laboratories to incorporate these new technologies into existing methods.

Charting a new agenda
The next agenda for the industry is to develop and validate a protozoa method for finished water. The fortunate aspect of developing this method is that the components used in Method 1623 can be applied to finished water. While fewer particles are associated with finished water than source water, treatment chemicals create different challenges for recovering oocysts and cysts from the matrix. Hundreds of liters of finished water can be collected on one filter allowing for the buildup of treatment chemicals such as polymers and alum on the filter material surface. The oocysts and cysts become embedded in this coagulated material and are exceedingly difficult to remove. Initial trials using pre-elution dispersants to break apart the particles and coagulants have shown promising results in trials on two types of finished water. Further evaluation is needed to determine if this method enhancement is universally applicable.

Research into adapting Method 1623 for recovery and detection of Crypto in sewage influent/effluent and biosolids is currently under way. The oocyst recovery rate in raw sewage is highly variable due to the presence of large particles and debris along with fats. Raw wastewaters are collected as grab or catch samples (a sample collected at a particular time as opposed to collection over a length of time). Incorporation of an internal positive control will be added to the sample prior to analysis that will serve as a guide to the expected recovery rate of the naturally occurring organisms. This internal control is a special preparation of stained Cryptosporidium that’s easily distinguishable from naturally occurring oocysts. Secondary and tertiary wastewater effluent matrices are similar to finished water so sample collection can be achieved using filtration. The problems associated with collecting a wastewater sample on a filter are the same as finished water. Treatment chemicals used to coagulate the particles and enhance settling create a coagulated muck that makes recovery of oocysts difficult. Validation studies for the sewage methods will be conducted by the end of the year.

With development and validation of Method 1623, some say accurate information is being obtained about the total number of organisms present in the environment. While this method is more sensitive than the ICR method, a method detection value (MDV) hasn’t been determined. Therefore, it’s difficult to say how sensitive the method is. Another issue to consider is that the detection step used in the method doesn’t give any information about the viability, or infectivity, of those organisms recovered. Only those organisms that are viable and infectious present a public health concern. Recent studies show that with minor modifications to Method 1623, cell culture techniques can distinguish infective cysts and oocysts. While such techniques are technically difficult and expensive—and only a few laboratories are set up to run the procedures—water testing’s future will likely include this piece of information for regulatory agencies and the drinking water suppliers to accurately assess treatment requirements and performance and determine public health risks.


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About the author
Randi M. McCuin is a senior microbiologist with Clancy Environmental Consultants Inc., of St. Albans, Vt. She has been with Clancy since 1994 and has been involved in the development of methods for recovery and detection of Giardia and Cryptosporidium in source water, drinking water and wastewater. She can be reached at (802) 527-2460 or email: [email protected].


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