[In-Plant Operations] Rapid Microbiology

Rapid microbiology test kits can be valuable resources for in-plant testing, as long as lab technicians take a few steps of validation prior to introducing a new test into plant routine. While the rapid test kit is created to be fairly simple and, by its very name, faster than traditional testing methods, "it is not necessarily as plug and play as people expect it to be," says Brad Stawick, president and technical director, Stawick Laboratory Management, Memphis, Tenn.

While agreeing that the rapid detection of microbial contaminants is critical for food safety, and noting the value of rapid methods which "make detection and identification faster, more convenient, more sensitive, and more specific than conventional assays – at least in theory," the publication, Rapid Methods for Detecting Foodborne Pathogens, from the Food and Drug Administration’s Center for Food Science and Nutrition (CFSAN) also encourages testers to select and evaluate rapid tests carefully before use. "Because of the complex designs and formats of these tests, coupled with the difficulties of testing foods, users must exercise caution when selecting rapid methods and to also evaluate these tests thoroughly, as some may be more suitable than others for distinct testing situations or for assaying certain types of food," states the 2001 publication, which is Appendix 1 of CFSAN’s Bacteriological Analytical Manual.

Among the evaluations that should be conducted prior to introducing a new rapid test into a plant’s routine are:

1. Manufacturer Validation – Before a lab uses a test kit, technicians should review the validation that has
been conducted by the manufacturer on the kit. This
will enable the lab to narrow the selection and determine
the best fit for its environment and products.

2. Plant and Product Validation – Once the selection is
narrowed, the plant needs to test the kit in its environment, running validation tests and ensuring that the
kit works as expected. "You need to do
an in-house validation to make sure
your dominant matrices are covered,"
Stawick says. "I think there’s a com-
mon misunderstanding that if a method is validated for one product type, it’s
validated for all product types." But, in reality, even within products of the same type, there are many variations, he says. For example, look through any grocery store and compare similar products from different manufactur-
ers. Whether you are comparing barbeque sauce, pizza or soup, you will find extensive variations in ingredients and production methods. "In theory, each variable complicates the issue,"
he says.

Evaluations of rapid methods do show that the tests vary in performance among different foods, states the CFSAN publication. "This can be attributed mostly to interference by food components, some of which can be especially troublesome for the technologies used in rapid methods." An example cited is that an ingredient could inhibit DNA hybridization or Taq polymerase, but have no effect on antigen-antibody interactions or the inverse could occur. "Since method efficiencies may be food dependent, it is advisable to perform comparative studies to ensure that a particular assay will be effective in the analysis of that food type," CFSAN notes.

Plants which produce single- or few-ingredient products will not need to do as much testing as those with more complicated, multi-ingredient products, says Stawick. For example a poultry processor need only conduct tests on a poultry matrix, whereas a pizza or frozen-dinner processor will have multiple ingredients as well as the final product against which the tests must be validated. "The complexity of the issue depends on what you are manufacturing." The more types of samples to be tested, the more factors the lab has to consider.

3. Method comparison – Labs also need
to take care if they are attempting to
compare quantitative results of differ-
ent test methods, Stawick says. "Why
don’t these numbers match?" he says
is a very common question, whereas the
answer is generally based on the fact that
the principle behind each test is differ-
ent, thus the quantitative result will
also vary. Instead of comparing meth-
ods against one another, tests need to
be judged against their own criteria
with the final analysis as positive or
negative results of pathogen pres-
ence.

4. "Ruggedness" testing – An additional
test labs should perform prior to the
first utilization of a new test is rugged-
ness testing. "How far can you stray off
the typical method and still get the
result that you expect," Stawick explains
the test as determining. This will pro-
vide parameters for the testing, as well
as showing its applicability to your
environment.

Stawick uses the example of a car engine to explain the concept. Say an automobile manufacturer is designing a new type of engine for fuel efficiency. While the engine is on the drawing board, one can’t completely predict how it will work until it is run in a real environment and put to the test in different situations. Even then, the engine will have different effects and efficiencies if used in different environments, e.g., a pick-up truck vs. a sub-compact. "The complexity of food really makes it difficult," Stawick says, coming back to the food industry, but the final analysis is actually quite simple, "If you make meat and cheese lasagna, test it on that, not on a chicken pot pie."

5. Result validation – Should rapid tests
indicate positive presence of the patho-
gen being tested, this is not to be taken
as a conclusive result, but as an indicator
of the need for further testing. While
the design of the rapid methods toward
detection of a single target makes them
ideal for quick screening of large num-
bers of food samples for the presence of
a particular pathogen or toxin, the
CFSAN publication states, "A posi-
tive result by a rapid method however,
is only regarded as presumptive and
must be confirmed by standard meth-
ods."

If the standard method begets a negative result, the rapid test result is generally determined to be a false positive with the specificity and sensitivity of the traditional tests taking precedence. But because traditional methods can also be less sensitive than some of the newer rapid tests, some plants will pull in a third method, or send a sample out to a contract laboratory, for confirmation of presence or absence of the pathogen. "It’s just a difficult situation," Stawick says.

6. Test Sensitivity – Testing methods can also vary in the level of analyte to which they test, with tests constantly gaining in sensitivity, and questions beginning to be asked as to how low should we go? If a product has one part per trillion, is it significant? Is it enough for a plant to test to parts per million?

When working with chemistry testing (such as pesticide, heavy metal, etc.), it can be a valid question. Say, for example, a product has one part per trillion pesticide residual. If the method being used tests to parts per million, the test will legitimately show no presence; if however the method tests to parts per trillion, it will show presence, and action will need to be taken.

However when dealing with microbial pathogens such as Salmonella, Stawick says, "according to FDA, any level of Salmonella is considered to be dangerous." Because the pathogens have the ability to grow from even minute amounts, one can legitimately argue that test sensitivity should continue to increase; and "parts per …" should go as low as conceivably possible. For example, if even a very low level of the pathogen exists in a product and the product is temperature abused at all, the pathogen is left to grow, and what was a barely detectable level can become a substantial level.

To ensure they are running the best possible tests, it is important that laboratory personnel stay up-to-date on the ever-emerging developments of the rapid testing. As is noted in the CFSAN publication, the Rapid Methods section differs from others in the manual in that the methods are not necessarily used by FDA and users are referred to test kit instructions for detailed protocol. "One reason for this departure is the incremental rate of change and innovation in rapid testing technology," the publication notes. "The best of these new techniques should be evaluated individually by user labs for their particular needs, and also collaboratively for possible adoption as official methods by the AOAC International."

"There’s hardly a day that goes by that you don’t see a press release on some new approach to pathogen detection," Stawick says. Whether it is a new take on an old method or a completely new method, "if you don’t stay up on it, you can be passed by." QA

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Product Guide: Rapid Microbiology Tests

Warnex Diagnostics www.warnex.ca 888-988-1888

Warnex Diagnostics offers a state-of-the-art DNA-based technology for the food industry. The WarnexTM Rapid Pathogen Detection System is a versatile detection and quantification platform, which uses real-time PCR technology combined with proprietary genetic markers and software, to rapidly and accurately determine the presence of pathogens in a sample. The system allows for the simultaneous detection of multiple pathogens and processing of samples within 3 to 48 hours, a significant improvement over traditional microbiology tests that require 5 to 7 days. Warnex also offers AOAC-validated detection kits for Salmonella spp., Listeria spp., Listeria monocytogenes, E. coli O157 and E. coli O157:H7, and a quantification kit for Campylobacter jejuni, C. coli and C. lari.

Microbiology International www.800EZmicro.com 800-396-4276

BBL CHROMagar is a selective medium for the isolation, differentiation and identification of microorganisms (Listeria, Salmonella, E.coli O157, Staph aureus) from food and environmental samples. CHROMagar has been validated by the AOAC Research Institute under the Performance Tested Methods Program as a single plate method for analysis when using FDA BAM, USDA FSIS, AOAC and ISO methods. Chromogenic media is growing in popularity as companies are finding the benefit of using a single plate method for classification.

Microgen latex agglutination kits for Salmonella, Listeria, E.coli O157:H7 offer a rapid alternative to traditional identification methods. Suspect colonies grown on agar media may be tested directly providing an early presumptive confirmation of genus prior to biochemical identification. This latex agglutination test has a very high specificity making it ideal for testing food, clinical, environmental and veterinary samples.

Microgen biochemical ID kits for species identification are easy to use, give clear distinction between positive and negative results for each substrate. These identification products are supported by the most comprehensive, up-to-date Taxa and comprehensive guidance notes for interpretation. Kits are available for species identification for the most commonly encountered organisms isolated by QC testing and research laboratories including gram negative bacilli E.coli, Salmonella, Yersinia, Shigella, etc., along with species identification kits for gram positive organisms i.e. Listeria, Bacillus, and Staphylococcus.

Charm Sciences www.charm.com 978.687.9200

Charm Sciences offers pre-operational rapid hygiene surveillance kits (< 20 seconds) based on ATP (adenosine triphosphate) detection. Available on the FireFly-2 and novaLUM, Charm’s PocketSwab Plus and WaterGiene offer real-time solutions to monitoring cleanliness and rinse water quality, allowing immediate corrective measures prior to product overlay. In addition, Allergiene, a highly sensitive ATP hygiene test designed to help prevent cross contamination of allergenic food soil from poorly cleaned process equipment, is available on the novaLUM. All Charm instruments come with software solutions featuring intuitive report management and dynamic graphing tools.