October 2003: Volume 45, Number 10
Detecting Waterborne Pathogens-- A Look at Past, Present and Future Approaches
by Kelly A. Reynolds, MSPH, Ph.D.
Human enteric pathogens are known to cause widespread waterborne disease in the United States, costing an estimated $20 billion a year in lost productivity. In the world, waterborne disease is thought to account for nearly 6,000 deaths per day, mostly in children. While we have a vast understanding of the variety of microbes in our environment and their association with disease, much progress is yet to be made.
Detection methods for microbes have come a long way since Anton van Leeuwenhoek first constructed a microscope and discovered the microbial world (circa 1680); however, about half of all drinking water outbreaks today are caused by unknown or unidentified agents. The viruses responsible for a significant portion of gastrointestinal illness in the United States, i.e., caliciviruses, can not be cultured in the laboratory. In addition, new strains of pathogens continue to emerge and documented microbial disease incidence is thought to be the tip of the iceberg with regard to true public health impact associated with microbial pathogens.
The difficulty in targeting a specific microbe as the causative agent of an outbreak or a disease is in part due to the lack of appropriate and effective methodologies for their detection. The inability to rapidly and effectively detect human enteric pathogens in drinking water has led to use of indicator organisms to monitor drinking water quality. This has limited research efforts to directly identify, characterize and evaluate risks as well as risk reduction approaches for a wide variety of disease-causing microbes.
So few pathogens, so much water
Enteric pathogens are comprised of three major groups--viruses, bacteria and protozoa. Each group is highly diverse with respect to size and physical and chemical characteristics. Conventional detection methods for identifying the presence of enteric pathogens in drinking water are often not suitable for routine monitoring due to their high cost, lengthy processing times or inadequate sensitivity. Sensitive detection of many enteric pathogens is necessary for public health and safety since as few as one infectious organism can cause disease in humans.
Typical microbial detection methods to evaluate drinking water sources begin with a filtration or concentration step aimed at isolating small concentrations of pathogens, i.e., one to 10 infectious units, from large volumes of water (hundreds to thousands of liters). Developing a universal filtration method suitable for efficient concentration of viruses, bacteria and protozoa alike has been challenging, with viruses ranging in size from 20 to 250 nanometers (.02 to .25 microns--m), bacteria from 0.2 to 5.0 m, and protozoa from 0.5 to 100 m.
Hollow fiber filters appear to be the most promising technology for concentration of a variety of pathogens from a water source. Using principles of ultrafiltration, the small pore sizes of hollow fiber filters are used to separate colloidal particles from a fluid stream based on molecular weight cut off (MWCO) values. Typical MWCO values range from 6,000 to 100,000 daltons. Tangential flow is used to pass the water sample over the filter surface to prevent clogging by suspending particles in the retained solution. Flow is maintained by spinning cartridges, stirred cells or other types of systems. An advantage to this crossflow circulation, as opposed to a flow through tortuous path filtration method, is that concentrated microbes aren’t compacted and subject to dessication as with conventional depth filters.
Water’s filtered…now what?
The concentration of hundreds of liters of water into less than 50 milliliters often results in the accumulation of compounds that inhibit to subsequent detection methodologies, requiring additional purification procedures to isolate target pathogens from background interference. The problem is that additional efforts to clean up the sample are invariably associated with a loss of the target pathogen. Efforts of purification must be carefully balanced against absolute pathogen recovery efficiencies.
Following filtration, there are a variety of options for the detection of specific pathogens including cultural methodologies, microscopy, immunochemical approaches and molecular methods. Each has their own particular advantages and disadvantages relative to specific pathogens. Cultural methods, for example, may be an appropriate method for detection of some bacteria, but are prohibitively time consuming and costly for virus analysis. No one cell line supports all enteric viruses and some are very slow growing (i.e., HAV, astroviruses) or produce questionable results in laboratory cultures. Others don’t grow at all. Likewise, protozoan pathogens and many bacterial pathogens are slow growing and require specific growth media, making cultural techniques an unlikely choice for a universal detection method.
Microscopy is quickly eliminated as a method for multiple pathogen detection as viruses are too small to be seen with standard scopes, requiring specialized electron microscopes. In addition, microscopy isn’t suitable for surveying a water concentrate for a low number of organisms since background interference would make this a tedious approach to pathogen monitoring.
Immumochemical methodologies have shown great promise for purifying target microbes from background interference. These immunoassays utilize receptor sites on the surface of microbes (antigens) that bind to host receptor sites (antibodies) to subsequently detect the microbe’s presence. Unfortunately, these methods aren’t sensitive enough for direct detection of pathogens from drinking water samples, requiring a minimum of 10,000 target microbes for reliable detection. When combined with culturable methods, however, immunoassays are useful for the detection of slow-growing and hard-to-detect pathogens. In addition, this method can be automated and suited to a variety of microbes, but has been criticized due to its inability to distinguish between infectious and non-infectious organisms.
A universal system
The best method for direct monitoring of drinking water for pathogens would provide a non-technical, automated, rapid technique for the detection, identification, enumeration and characterization of the target organism. Many scientists agree that a molecular-based approach shows the greatest promise for the development of a unified system for multiple waterborne pathogen detection.
In the mid-to-late 80s, polymerase chain reaction (PCR) was coming onto the microbiology scene. PCR is an amplification method for the detection of microbes utilizing their nucleic acid (gene sequences). Because nucleic acid sequences are like fingerprints for a particular type of microbe, this method can be designed to detect and identify specific targets. The method is very sensitive, theoretically detecting a single infectious particle. The problem is the method can produce false negative results due to the presence of inhibitory compounds, or false positive results, if not carefully optimized. Furthermore, molecular methods can detect non-infectious organisms and bring into question the public health significance of a PCR positive result.
Given these limitations, research previously has been focused on optimization of the PCR reaction to overcome effects of inhibitory compounds. Methods were also developed to combine cultural and molecular methodologies. Detection of viable enteric viruses, for example, was drastically improved by the use of combined cultural and molecular approaches. By placing viruses on mammalian cell culture for a limited period of time, a cultural amplification step takes place that’s subsequently detected by the rapid and specific PCR methodology. The overall method can be performed in hours compared to weeks with conventional cultural methods alone.
Although newer molecular methods can be problematic and produce questionable results relating to uncertain risks, they frequently serve as a rapid screening tool to be later verified by traditional methods whenever a red flag goes up. Despite its limitations, PCR methods have been successfully used to identify the causative agent for waterborne outbreaks when no agent could otherwise be cultured.
A look ahead to approaches
The goal of methods development for pathogen detection must include an in-line, sensitive approach. Promising methods utilizing automation, real-time detection and molecular technology have been used in clinical and industrial settings, and are being adapted to environmental sample monitoring.
Microarrays are a rapidly evolving molecular method that appears to have applications for water monitoring. Like PCR, the technology is based on the Watson and Crick model of double-stranded DNA/RNA molecules that bond (hybridize) together in a defined order. Microarrays utilize gene chips, i.e., probes, anchored to glass or nylon slides. Each spot on a microarray is intended to hybridize to a specific target sequence. Using automated robotics, tens of thousands of spots can be placed on a single array. These high-density throughput probes can be used for detection of multiple target sequences and have been used to directly detect RNA without a PCR amplification step. Experts are trying to develop the technology with real-time monitoring approaches for a multitude of microbial pathogens in water.
Perhaps the biggest push for the development of rapid monitoring and diagnostics for pathogens in drinking water is the potential for bioterrorism events. Currently, a great challenge in the response to a bioterrorism attack is how to identify that an event has occurred. To do this, the environment needs to be routinely monitored for a wide variety of pathogens.
Since the recent anthrax attack, the U.S. Postal Service has initiated research on the manufacture of a biohazard detection system that rapidly identifies biological pathogens, i.e., Cepheid’s GeneXpert. These systems are designed to detect trace amounts of anthrax DNA and other airborne agents. Using real-time PCR technology and automated analysis instrumentation, new systems can detect the presence of a target pathogen in 30 minutes or less.
PCR systems have been developed that are portable--i.e., Idaho Technology Inc.’s Ruggedized Advanced Pathogen Identification Device--allowing for direct monitoring of a site without the need to collect and transport samples back to the laboratory. This saves precious time that could be used for response and containment of a contamination event.
A low-cost, portable, DNA chip-based system is also in the works via Integrated Nano-Technologies’ BioDetect. Targeting possible biological warfare agents such as anthrax, smallpox and SARS, this system can provide accurate results in minutes and is extremely lightweight (12 pounds). The system utilizes microchip technology to target hundreds of pathogens simultaneously.
While these recent advances in technology are promising, it shouldn’t be assumed real-time applications are foolproof. Many problems remain to be worked out such as background interferences, false-positive and false-negative signals, and the issue of the need for large volume testing that’s an inherent problem in drinking water analysis, particularly for municipal systems.
Although new molecular methods have been developed for the detection of human pathogens in drinking and source waters that surpass traditional approaches, many have yet to be evaluated in environmental microbiology where natural components can interfere with method efficacy and sensitivity. The problem is many new methods, while an improvement over conventional methodology, aren’t widely applied or universally accepted. Therefore, their meaning from a risk assessment approach isn’t known. To expand the acceptance and increase development of new methodologies, methods must continue to be applied to gather data for use in occurrence and exposure studies as well as risk assessment modeling so consistent validation protocols can be developed. The future promises to be interesting.
1. WHO/UNICEF, “Global water supply and sanitation assessment 2000 report,” Water For People, 2000: www.water4people.org/default.htm
2. Straub, T.M., and D.P. Chandler, “Towards a unified system for detecting waterborne pathogens,” Journal of Microbiological Methods, 53: 185-197, 2003.
3. Reynolds, K.A., et al., “Detection of infectious enteroviruses by an integrated cell culture-PCR procedure,” Applied Environmental Microbiology, 62: 1424-1427, 1996.
4. Frederickson, R.M., “Building biohazard detectors,” Bio-IT World (online), 2003: www.bio-itworld.com/archive/equipped/072103.html
About the author
Dr. Kelly A. Reynolds is a research scientist at the University of Arizona with a focus on development of rapid methods for detecting human pathogenic viruses in drinking water. She holds a master of science degree in public health (MSPH) from the University of South Florida and doctorate in microbiology from the University of Arizona. Reynolds has also been a member of the WC&P Technical Review Committee since 1997.
Although newer molecular methods can be problematic and produce questionable results relating to uncertain risks, they frequently serve as a rapid screening tool to be later verified by traditional methods...