Grantee Research Project Results
Final Report: Portable System for On-Site Legionella Detection
EPA Contract Number: 68D98125Title: Portable System for On-Site Legionella Detection
Investigators: Fernandez, Salvador M.
Small Business: Ciencia Inc.
EPA Contact:
Phase: I
Project Period: September 1, 1998 through March 1, 1999
Project Amount: $70,000
RFA: Small Business Innovation Research (SBIR) - Phase I (1998) RFA Text | Recipients Lists
Research Category: Ecological Indicators/Assessment/Restoration , SBIR - Monitoring , Small Business Innovation Research (SBIR)
Summary/Accomplishments (Outputs/Outcomes):
The long-range objective of this program is to develop a portable system for rapid, on site, quantitative testing for Legionella pneumophila and other environmental pathogens. The objectives of the Phase I effort is to demonstrate the feasibility of a fluoroimmunoassay approach based on the use of novel fluorescent labels and an LED-based (light-emitting diode) detection system.Legionella pneumophila is the bacterium associated with Legionnaires disease and Pontiac fever. There are at least 48 species of the Genus Legionella with more than 51 serogroups identified to date. L. pneumophila can be grouped into at least 14 serological variants. However, L. pneumophila serovar 1, the cause of the original outbreak is still the most important disease agent of the group.
Respiratory transmission of this organism can lead to infection. Patients may experience fever, chills, and may develop severe pneumonia, which is not responsive to penicillins or aminoglycosydes. Legionnaires' disease also has the potential to spread into other organ systems such as the gastrointestinal tract and the central nervous system. Accordingly, patients with advanced infection may experience diarrhea, nausea, disorientation, confusion and death.
Legionella are widely distributed in our environment. They are found in rivers, lakes and ponds, hot and cold water traps, hot water tanks, water in air conditioning cooling towers and evaporative condensers, and soil at excavation sites. When contaminated water is aerosolized, the risk of human infection is greatly increased. Thus, if man-made water systems produce jets, sprays or mists, as with cooling towers, showers and some types of humidifiers, it is important to minimize the chances of Legionella colonizing the water reservoirs, storage tanks and other aquatic systems serving them. Cooling towers are of particular importance because their operating temperatures are at an optimum level for Legionella growth, they are designed to aerosolize the water and they are easily and frequently contaminated by wind-blown dusts and soil particles which can carry with them disease-producing microorganisms including L. pneumophila. Outbreaks of legionellosis have been traced to a number of water sources including Legionella-containing water in cooling towers and air conditioners, hot tubs, showerheads, public fountains , and even a supermarket vegetable-misting machine.
The presence of L. pneumophila in building water systems is of prime importance to engineers, building managers and hygienists. The organisms can be controlled in such systems by the application of biocides and their detection and identification plays a vital role both in initial assessment of the water system and subsequent treatment effectiveness and ongoing water quality monitoring.
Water from cooling towers, spray-type humidifiers and other air conditioning associated equipment present the greatest degree of risk. Sound management of these systems requires that a routine sampling procedure from their water storage reservoirs and from other water systems be established. Since the average life cycle of L. pneumophila under the favorable conditions supplied by such systems is about three weeks, a once-a-month sampling frequency has been recommended. However, laboratory culture methods to isolate and identify Legionella take from 4 to 14 days to provide results. In some cases, it takes more than two weeks for most colonies to appear on the media. Clearly, a test turnaround time that is commensurate with the life cycle of the organism being tested is far from ideal, and of marginal adequacy for building health monitoring.
Current alternatives to culture detection methods such as polymerase chain reaction assays (PCR) and direct fluorescent antibody (DFA) staining also require that samples be sent to a laboratory for analysis. Although these assay procedures may be faster than culture methods, they still suffer from inherently slow turnaround times because of the need for off-site analysis. They are also expensive.
An effective monitoring protocol should provide the capability to inexpensively test water samples for L. pneumophila on site immediately prior to routine cleaning and maintenance of cooling towers, humidifiers and other water systems. If it is found present, then the necessary water treatment can be performed and its effectiveness promptly verified. Only then can maintenance regimes and water treatment protocols be established on the basis of need rather than on guesswork. This is not possible with current test methods, which are laboratory based and do not permit rapid on site testing. Our technical approach rests on the use of novel fluorescent markers to make fluorescent anti-Legionella antibody conjugates, and a low-cost, compact, rugged, low power detection system based in light-emitting diodes (LED). These two elements are combined to develop a portable direct immunoassay system of high sensitivity.
We developed a conjugation procedure and made conjugates that stained Legionella brightly and specifically as demonstrated by microscopic examination of stained bacteria in a fluorescence microscope. We also built a prototype of an LED-based fluorimeter and developed a filtration assay protocol. Our results demonstrated that the proposed dye provides over three orders of magnitude greater sensitivity that fluorescein, the dye currently used in the DFA Legionella assay. In terms of Legionella minimum detection limit, this translates to 102 bacteria per mL. This is the same detection limit that is now possible with the laboratory-based DFA technique, which requires a fluorescence microscope and bacterial counting by a trained technician.
Our results have successfully demonstrated the feasibility of the proposed novel approach for the development of a low-cost, rugged, portable system for rapid on-site detection of Legionella
Supplemental Keywords:
RFA, Scientific Discipline, Water, Ecosystem Protection/Environmental Exposure & Risk, Monitoring/Modeling, Analytical Chemistry, Microbiology, Engineering, Chemistry, & Physics, environmental monitoring, microbial monitoring, pathogens, field portable monitoring, field portable systems, bacteria monitoring, field monitoring, municipal wastewater, biomonitoring, fluorescence detection, Ligionella detection, indoor air, measurement, innovative technologies, flourescence assay, microbial flora, atmospheric chemistrySBIR Phase II:
Portable System for On-Site Detection of Legionella | 1998 Progress Report | Final ReportThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.