Medical Device Link.

Originally Published IVD Technology June 2001

Electroimmunoassay technology for food-borne-pathogen detection

Sharon Brunelle

A technology for the detection of pathogens in many types of food products may also show promise for use in other IVD tests.

Sharon Brunelle, PhD, is the chief scientific officer at Molecular Circuitry (King of Prussia, PA). She is responsible for overseeing all matters related to the company's core functions in research and development as well as creating product development strategies. Dr. Brunelle can be reached at sbrunelle@molc.net.

In fact, in 1999, the Centers for Disease Control and Prevention (CDC; Atlanta) reported that each year at least 76 million people in the United States become sick from food-borne pathogens.¹ Such illnesses result in 325,000 hospitalizations and 5000 deaths annually.

Major food-borne pathogens include Salmonella, Listeria monocytogenes, Campylobacter, and Escherichia coli O157:H7 (see Table I). According to estimates by the United States Department of Agriculture (USDA), the medical costs and losses of productivity resulting from food-borne illnesses stemming from these major food pathogens range between $6.5 billion and $34.9 billion annually. In an effort to reduce the likelihood of contaminated food products entering the nation's food supply, FDA and the Food Safety Inspection Service (FSIS), a division of USDA, have devised prevention programs aimed especially at these four pathogens. Following the 1993 outbreak of E. coli O157:H7 in the United States, for instance, USDA responded by mandating the inclusion of safe-handling statements in the labeling of all raw and partially cooked meat and poultry products.

Processing Facility Regulations

Since that time, USDA and FDA regulations imposed on the food-processing industry include requirements for science-based testing for pathogens in food products. Current FSIS regulations for the food-processing industry include mandatory testing programs for E. coli O157:H7 in raw beef and for Salmonella in raw beef and poultry as well as eggs. Under these regulations, if a food sample routinely tested by FSIS is found positive for Salmonella, E. coli, or L. monocytogenes, the food-production plant where the sample was processed is required to take preventive and corrective actions. Such actions may include destroying the contaminated lot of product or altering the plant's processes. Unlike FDA, USDA cannot mandate a product recall or plant closure. However, USDA can withdraw the mandatory inspector from the premises, effectively closing a plant's operations.

FSIS has recently published a proposed rule that would change existing practice by requiring processors of all ready-to-eat and partially heat-treated meat and poultry items to ensure that processing conditions are sufficient to reduce pathogen levels by specified amounts.² The USDA has statistically determined these reduction amounts to theoretically result in unadulterated product.

The proposed rule would require all inspected establishments that produce ready-to-eat meat and poultry products, but have not developed and implemented postlethality treatment controls for L. monocytogenes in their hazard analysis and critical control points (HACCP) plans, to test food contact surfaces for Listeria. In preparation for the finalization of this rule, industry has already begun to adopt such HACCP programs, which are designed to monitor the critical steps or areas in the processing of food.

HACCP programs enable federally inspected establishments to take preventive and corrective measures at each stage of the food-production process where food-safety hazards are known to occur. Such monitoring includes the environmental screening of machines and surfaces for indicator organisms such as the genus Listeria. Environmental screening for the presence of Listeria genus organisms is intended only as an indication of the level of sanitation in the processing area, as L. monocytogenes is the only species of Listeria that is pathogenic. Food ingredients intended for processing, however, are tested for specific pathogens, such as L. monocytogenes, as part of a good HACCP plan. Although the implementation of HACCP reduces the risk of pathogen contamination of food products, it does not eliminate the need to test final product prior to shipping.

In the United States, the HACCP concept is now being broadly implemented in the food-service industry, which includes retail establishments such as restaurants, caterers, cafeterias, and supermarkets. At least half of the states have passed legislation requiring HACCP programs, or have legislation pending to do so.

Food-Pathogen Detection Market

In 1999, the U.S. food industry performed at least 144.3 million microbiological tests.³ Of these, 23.5 million (16.3%) were pathogen-specific tests. This figure represents a 23.3% increase over 1998 estimates. The 1999 U.S. market size for food-safety tests is estimated at $53.4 million, representing approximately 30% of the total world market.

Food-pathogen testing is performed in a variety of locations. These include government laboratories, reference laboratories, centralized corporate laboratories, and on-site laboratories at food-processing plants.

Over the past few years, there has been a shift toward performing more pathogen testing on-site in food-processing plants. This shift can be attributed to several factors. One reason is that increased testing volumes, largely due to the implementation of HACCP and other regulations, have made it more economical for many processing plants to set up laboratories on-site rather than continue to send samples to outside reference or corporate laboratories.

Another reason for the growth of on-site testing is that results are available more quickly. This is important because USDA directives require that raw ground beef products be held in inventory until E. coli O157:H7 test results are obtained. All other products can be shipped once a sample has been retained for testing. Testing on-site allows results to be obtained sooner, shortening the amount of time ground beef products must be retained in inventory (and lowering the storage costs), and providing a more rapid response time if a recall of other types of products is warranted.

Trends in Testing Methods

Science-based pathogen-specific tests can be divided into the categories of traditional and rapid methods. Traditional microbiological methods involve enriching the food sample and performing various media-based metabolic tests (agar plates or slants). These typically require 3–7 days to obtain a result.

Borrowing from clinical laboratory methods, rapid-screening tests based on immunochemical or nucleic acid technologies have been developed for food testing. These tests can provide results in 8 to 48 hours. However, results from these screening tests are considered presumptive by the USDA, which requires an isolated organism as proof of contamination.

Currently, the number of tests being performed is divided evenly between traditional tests and rapid screening methods.³ This is true for both the U.S. and worldwide markets. Industry experts expect that, as regulations pertaining to pathogen testing continue to be adopted and the amount of testing increases, the shift toward rapid-screening methods will continue.

The three types of rapid-test methods are manual, semiautomated, and fully automated. Manual rapid-test methods include lateral-flow tests and dipstick immunoassays. Semiautomated methods include ELISA tests and enzyme-linked hybridization tests. Fully automated systems include instrumentation to perform either immunoassays or PCR methodologies. Table II shows examples of product technologies marketed in this field.

Laboratories that perform low-volume testing generally use the manual tests, whereas medium- and high-volume testing labs use the semi- or fully automated tests. Manual tests have subjective visual readouts dependent on the interpretation of the user whereas semi- and fully automated tests provide objective measurement-based results. The objective measurements can be optical or electrical.

Methodology

Typically, food or beverage samples are tested by placing 25 g of randomly sampled product into 225 ml of enrichment medium appropriate for the growth of the target organism.^4,5 Enrichment protocols may include a single selective medium or up to three media conditions in order to achieve recovery of injured organisms (very important for processed foods), high growth, and maximum selectivity (suppression of nontarget organisms).

In an effort to shorten the overall time to result and to enhance selectivity, new proprietary medium formulations and immunocapture techniques have been developed. Immunocapture is the selective concentration of target organisms using antibody-coated magnetic beads or other devices to selectively trap and transfer the organisms to fresh broth. These methods can be effective for reducing background flora and eliminating interfering food substances, but are labor intensive and therefore not well suited for high-volume users.

Once a sample has been enriched, an aliquot is typically removed and processed (usually boiled) prior to testing by immunoassay. Some lateral-flow tests are compatible with unprocessed samples (i.e., live bacteria), thus eliminating one step for the user.

Overview of Electroimmunoassay Technology

Figure 1. Electroimmunoassay technology is composed of a circuit with a capture antibody attached to the solid surface in the area of the electrode gap.

Electroimmunoassays couple specific antibody-antigen binding to the production of an electrical signal. The technology is comprised of a circuit with a capture antibody attached to the solid surface in the area of the electrode gap (see Figure 1). Upon addition of sample, the target antigen binds to the capture antibody. In the next step, a colloidal gold–labeled detection antibody is bound, creating a capture-target-detector sandwich. The final step is the deposition of silver ions onto the colloidal gold, which produces a conductive silver bridge, closing the circuit and resulting in a measured drop in resistance. This detection technology can also be applied to nucleic acid hybridization assays.

Figure 2. The Detex electroimmunoassay biosensor system consists of the MC-18 immunoassay instrument and disposable pathogen-detection kits for various target pathogens.

The MC-18 can perform 27 tests per run, divided into nine sectors of three tests each. The system is multiparametric, meaning that any combination of pathogen tests can be performed simultaneously. For example, one sector can be loaded for E. coli O157 testing and the next sector for Salmonella testing.

The system is also capable of duplex testing, meaning that it can conduct two tests in one cartridge. For example, one sample can simultaneously be tested for both Salmonella and E. coli O157. This is achieved by the addition of a second circuit to the cartridge. Dual enrichment media formulations compatible with the duplex tests are also being developed by the company.

The system is fully automated, enabling the user to complete the following steps.

• Enrich the food product in the appropriate broth(s).
• Heat-inactivate a sample of the enriched broth.
• Load the appropriate reagent packs and cartridges into the carousel.
• Add a 150 µl aliquot to the cartridge.

Using a bar code scanner, the MC-18 instrument scans the reagent packs and cartridges to ensure that matching components have been loaded into each sector. External bar code reading capability is being developed to enable the user to scan bar coded samples.

The system then performs all remaining steps of the assays, including reagent dispensing, aspirating, and washing. After the final step, the instrument measures the resistance of the circuit in each cartridge and determines a positive or negative output. The test results are automatically saved to a floppy disk in an Excel-compatible format and can be sent directly to an attached printer.

Alternatively, a PC can be linked directly to the instrument for automatic downloading of data. This data management feature eliminates data transcription errors, a potential problem associated with manual and some semiautomated methods, and reduces the potential for shipping or withholding product erroneously.

Assay Development

One of the most difficult aspects of developing pathogen-specific assays for the food industry is the wide variety of potential sample matrices. For example, a Salmonella test method should be compatible with such diverse matrices as shellfish, meats, ice cream, egg products, spices, and fruit juices.

The Association of Official Analytical Chemists International (AOAC) recommends 14 different food groups to be included in validation studies for Salmonella tests. The foods cover a wide range of acidity levels, salt concentrations, fat and moisture content, and competing bacterial flora.

As a result, food type-specific enrichment protocols are often necessary. Processed foods, for example, generally have a lower microbial load than raw foods, but recovery of organisms can be difficult due to heat injury. The enrichment protocols for processed foods, therefore, usually involve less selective and less stressful conditions than for raw foods. Some food components can interfere directly with diagnostic methods. For example, it has been reported that casein hydrolysate, calcium ions, and certain components of some enrichment broths are inhibitory for PCR methods.⁶

Another difficult aspect is the specificity of detection reagents. For example, more than 2400 serotypes of Salmonella have been identified to date. The challenge to the assay developer is to identify a target or group of target antigens or nucleic acid sequences that would allow detection of all Salmonella serotypes without false positives because of the presence of closely related gram-negative bacteria such as Citrobacter.

Of particular difficulty within the Salmonella genus are the S. arizonae subspecies, which were at one time considered a distinct genus, but have since been classified within the Salmonella genus. Similarly, the antigenic detection of L. monocytogenes is difficult because of overlapping antigenicity with other nonpathogenic species of the genus Listeria. Recombinant DNA approaches coupled with monoclonal antibody generation can help to target individual epitopes of interest.

Assay Validation

Neither FDA nor USDA regulates diagnostic products for industrial microbiology. Instead, AOAC, an independent nongovernmental organization, oversees validation studies that are carried out to compare new diagnostic products to the current standard FDA or USDA/FSIS protocols.^4,5 To obtain AOAC certification, a test must be shown to be equivalent to or better than the standard methods when tested both internally and at independent external laboratories.

The method comparisons are carried out with all of the food groups that will be claimed and involve spiking foods at low (1 to 10 colony-forming units [CFU] per 25 g) and high (10 to 50 CFU per 25 g) levels of microorganisms, as well as testing uninoculated foods. The spiked foods are typically stored at 4°C for up to 72 hours before beginning the enrichment processes in order to stress the organisms, thereby testing the new method under challenging conditions. In addition to testing food matrices, extensive testing of bacterial species and strains, including target and nontarget organisms, is conducted to evaluate inclusivity (the industry equivalent of clinical sensitivity) and exclusivity (the industry equivalent of clinical specificity) of the method.

Other testing generally includes shelf-life testing of the kit and ruggedness testing of the method. Ruggedness testing attempts to address the effect of potential errors that could be introduced by the user such as incorrect sample volumes.

AOAC certification is not required by any U.S. regulatory agency. However, many food-testing labs, especially smaller labs that are already overworked, require AOAC certification in lieu of extensive validation efforts that would otherwise be required on their part.

Electroimmunoassay Performance

The Detex E. coli O157 pathogen detection kit was tested for sensitivity, specificity, and repeatability compared with the USDA/FSIS protocol in accordance with AOAC requirements for raw meat and poultry.⁷ This testing was carried out using a 24-hour protocol, which requires enrichment of raw meat samples (25 g) in modified E. coli broth (225 ml) at 37°C for 20–24 hours, followed by boiling or autoclaving. An aliquot (150 µl) of the heat-inactivated sample is then added to the cartridge and the assay is initiated on the MC-18 instrument. The results are summarized in Table III.

Some false positives randomly occurred, resulting in a specificity of only 90%. However, the organisms giving rise to the false-positive results were not consistent between the raw beef and the raw poultry matrices. It was determined that the false-positive results were due, in fact, to the presence of dried salts and other materials at the level of the meniscus on the resistor sheet, causing a short circuit. The problem was resolved by applying a dielectric coating over the electrode traces in the area of the meniscus.

Repeatability studies were carried out by inoculating food matrices at several levels as described above and testing 20 replicate samples at each level. Both in-house and external testing involved inoculation at levels of less than or equal to 1 organism per gram of food. In-house testing was performed on raw beef and raw chicken while external testing was performed on raw turkey (see Table IV).

The Detex E. coli O157 electroimmunoassay demonstrates good agreement with the USDA/FSIS reference method. There was 96% agreement from a total of 394 positive and negative samples evaluated with a sensitivity of less than 0.3 CFU/g of raw meat. The performance characteristics of the E. coli O157 electroimmunoassay are very competitive with those of other methodologies in the market. Validation of an 8-hour protocol using proprietary enrichment media is currently under way.

Future Applications of Electroimmunoassay Technology

As an immunoassay platform, the Detex system menu can be expanded to include tests for other antigenic targets for various markets. Alternatively, the electrical detection concept can be combined with molecular probes for carrying out hybridization assays, thus expanding the utility of the current platform with some modest changes such as temperature control.

Technology research is under way to employ the described electrical detection technology in an ultrasensitive quantitative biosensor device that would have broad applications in industrial and clinical diagnostics. There are reports of optical biosensor developments for food-borne pathogen detection.⁸ The ideal device would be an ultrasensitive in-line sensor that could quantitate total bacterial load as well as specific pathogen levels.

References

1. PS Mead et al., "Food-Related Illness and Death in the United States," Emerging Infectious Diseases 5, no. 5 (1999): 607–625.

2. Federal Register 66 FR:12590, February 27, 2001.

3. Pathogen Testing in the U.S. Food Industry (Woodstock, VT: Strategic Consulting, 2000).

4. Bacteriological Analytical Manual, 8th ed. (Gaithersburg, MD: Association of Official Analytical Chemists International, 1998).

5. Microbiology Laboratory Guidebook, 3rd ed. (Washington, DC: United States Department of Agriculture, Food Safety Inspection Service, 1998).

6. TJ Smith et al., "Molecular Diagnostics in Food Safety: Rapid Detection of Food-Borne Pathogens," Irish Journal of Agricultural and Food Research 39 (2000): 309–319.

7. YM Henry et al., "Detex for Detection of E. coli O157 in Raw Ground Beef and Raw Ground Poultry," Journal of AOAC International (forthcoming).

8. PJ O'Connell et al., "Biosensors for Food Analysis," Irish Journal of Agricultural and Food Research 39 (2000): 321–329.

Photo Courtesy Molecular Circuitry Inc.

Copyright ©2001 IVD Technology