Grantee Research Project Results

Final Report: A Portable Flow Cytometer Suitable for the Rapid Detection of Adenovirus in Wastewater

EPA Contract Number: 68D02026
Title: A Portable Flow Cytometer Suitable for the Rapid Detection of Adenovirus in Wastewater
Investigators: Kulaga, Henrietta
Small Business: GEOMET Technologies Inc.
EPA Contact: Richards, April
Phase: I
Project Period: April 1, 2002 through September 1, 2002
Project Amount: $70,000
RFA: Small Business Innovation Research (SBIR) - Phase I (2002) RFA Text |  Recipients Lists
Research Category: Ecological Indicators/Assessment/Restoration , SBIR - Monitoring , Small Business Innovation Research (SBIR)

Description:

The goal of this Phase I research project was to develop a portable system for the real-time detection and quantification of adenovirus in wastewater. The system is based on a small, portable flow cytometer that was developed and is being marketed by Becton Dickinson Biosciences under the trade name FACSCount™, for use in selected medical and research applications. Unlike previous efforts to apply flow cytometry to the testing of wastewater, the system used for this work is based on a capture bead assay that examines antibody-coated fluorescent microspheres that react with adenovirus.

Flow cytometry is a form of spectroscopy that interrogates individual members of a sample population for size, shape, biological, and chemical properties, thus making it a potential tool for determining the concentration of specific particles in a heterogeneous sample. Cells or fragments reacted with a reagent blend that is specific to the target biologic are carried by a fluid stream through a focus of exciting light. The distribution and intensity of the scattered light and fluorescence imparted by fluorescent probes generates both a scatter and fluorescence fingerprint that is interpreted by an internal computer that determines the presence and concentration of the target biologic. The key to using flow cytometry for this purpose is the development of reagents and software that are unique to adenovirus in wastewater.

Summary/Accomplishments (Outputs/Outcomes):

GEOMET Technologies, Inc., successfully developed the specific reagents and software to identify adenovirus in water. The Phase I research objectives were met and exceeded. Phase I had three objectives: (1) development of the reagent blend and software modifications that are unique to adenovirus, (2) confirmation that the system's lower detection limit is adequate for adenovirus analysis, and (3) evaluation of the impact of interfering materials on the analysis. The quality assurance objective for Phase I (as stated in the proposal) was 105 adenovirus plaque-forming units/mL¹ within 15 minutes. This time criterion was successfully met in all cases.

These results were obtained for adenovirus-spiked clean water and virally spiked water to which the following interferents had been individually added: Bacillus subtilis, Escherichia coli, and Pseudomonas aerogenosa. As a final indicator of the method's ability to work in real wastewater, the tests were repeated in water spiked with raw sewage that was obtained from the Washington Suburban Sanitary Commission. The sewage was tested, as received, to verify that it was free of adenovirus. Adenovirus was clearly identified in all of the spiked samples at a concentration of 100 adenovirus counts/mL and at higher concentrations. During some of the tests, adenovirus was detected at levels as low as 5x10^-4 counts/mL; however, at these low levels the interferents became a problem and the measurements could not be consistently confirmed. Results at the lower levels did indicate that more advanced statistical analysis algorithms in the FACSCount™ software could allow GEOMET Technologies, Inc., to lower the method's sensitivity to such lower levels. Phase II will incorporate advanced statistical methods.

These extremely low detection limits are possible because this method actually detects a shift in the measured parameters that is caused by antibody-coated capture microspheres that are coated with a secondary detector antibody that is sensitive to adenovirus, rather than direct immunofluoresence of adenovirus itself. Adenovirus binding to microspheres creates a resolvable particle; conjugated detector antibody binding amplifies the reaction.

Discussions with various researchers in the field, including within the U.S. Environmental Protection Agency (EPA) and in various regional water research consortia indicated that current methodologies detect adenovirus at concentrations on the order of 106 counts/mL, and even at these concentrations, they are subject to interference by other organic compounds.

The following tasks also were completed successfully:

• Developed an analysis "attractor" for identification of adenovirus in water.

• Identified workable sample stabilizers.

• Identified hardware and software changes that are needed to modify the FACSCount™ for use by a minimally trained operator to test for adenovirus in wastewater. The changes appear to be readily implementable.

• Developed the design for a simple, easy-to-use field sampling and analysis kit.

• Reworked the business plan (with review and input by Foresight) to incorporate the information obtained during the Phase I project. The business plan still indicates profitable operation within 2 years of the start of Phase II
  

¹The term commonly used in the field for the concentration of the target biologic is "plaque-forming units" or "oocots" per mL. For clarity in this report, which is expected to be read mainly by scientists and engineers with experience in the environmental field rather than in the field of biological assay, the term "counts per mL" or "adenovirus counts/mL" is used.

Conclusions:

In summary, the flow cytometric capture bead assay system proved to be highly selective for adenovirus. Common interferents at high levels, including sewage, did not impair the assay's lower detection limit. Furthermore, the method discriminated between the target material and interferents even in the presence of extraneous fluorescent dyes. Water samples, especially in the vicinity of refineries, often contain fluorescent contaminants.

Discussions also were held with EPA's Office of Water and National Exposure Research Laboratory in Cincinnati, OH, regarding use of the method for EPA's purposes. The results also were discussed with an individual from the Southern California Coastal Water Research Project and with Dr. Alben of the National Water Quality Monitoring Council. Both groups are following and encouraging research on rapid bioanalytical methods that can be used to quickly determine the safety of beach waters for swimming. Discussions with these groups indicated that the results were achieving lower detection and selectivity in the presence of serious intereferents than have been encountered elsewhere. These and numerous other discussions were very fruitful. GEOMET Technologies, Inc., concluded from these discussions that the results are of programmatic interest to these offices.

Supplemental Keywords:

wastewater, cytometer, adenovirus, assay, fluorescence, antibody-coated fluorescent microspheres, FACSCount™ software, real-time detection, Bacillus subtilis, Escherichia coli, Pseudomonas aerogenosa, SBIR, RFA, Scientific Discipline, Ecosystem Protection/Environmental Exposure & Risk, Water, Biology, Engineering, Chemistry, & Physics, Analytical Chemistry, Wastewater, Monitoring/Modeling, Arsenic, Ecology and Ecosystems, Environmental Engineering, Environmental Monitoring, contaminant transport, field detection, portable flow cytometer, water quality, field portable monitoring, field portable systems, antibodies, detection system, monitoring, aqueous waste stream, real-time monitoring, field monitoring, water contaminants, contaminants, wastewater treatment, municipal sewers, adenovirus, biomonitoring, stormwater, risk management