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From: Food Quality & Safety magazine, August/September 2013

Safeguarding Against False Test Results

by Maybelle Cowan-Lincoln

Safeguarding Against False Results

The larger the area we source our food from, the more we encounter the threat posed by a microscopic enemy: Foodborne pathogens. Good safety practices demand stringent, broad-spectrum testing to identify potentially dangerous microbes, but are the current testing products and procedures doing an adequate job? According to a recent report from the American Proficiency Institute, an independent agency that measures the accuracy of laboratories, there is significant room for improvement. New technology and new iterations of existing technologies hope to fill that need.

The food supply is becoming more and more global. Much of the produce, meat, and seafood found in U.S. markets and restaurants come from other countries. In fact, more than half of the food consumed on the planet is eaten in a different geography than it is grown or produced in. This opens our food supply to not only pathogens found locally, but also to microorganisms from all over the world.

Problematic Pathogens

Two of the most problematic pathogens are Salmonella and Campylobacter. As few as one to 10 Salmonella cells can cause disease, while 1,000 Campylobacter cells in contaminated poultry, raw milk, or produce can make a consumer ill. Of course, cooking does eliminate these pathogens, but food may still be handled between the time it is cooked and when it is served, opening up the possibility of bacteria being introduced by food handlers or servers.

Salmonella is the leading cause of foodborne illness according to FoodNet, the Centers for Disease Control and Prevention system for tracking foodborne infections. The number of Salmonella infections has remained relatively steady since 1996; however, there has been a shift in the strains sickening consumers. Wendy Lauer, senior product manager of Bio-Rad Laboratories, a San Francisco-based lab equipment provider, states, “Organisms continue to change and adapt. We have seen an increase in antibiotic-resistant Salmonella, for example. As a provider of solutions to the food industry, we have scientists working hard to keep up with these changes.” Infections from the most common strain have decreased, while illnesses caused by rising new strains, especially antibiotic-resistant strains, have increased. Infections from Campylobacter, the pathogen responsible for the third largest number of foodborne infections, are up 14 percent from the period between 2006 and 2008.

Mistakes in the Lab?

In May 2013, a report issued by the American Proficiency Institute and presented at the 113th General Meeting of the American Society for Microbiology revealed that over the past 14 years, the laboratories it has tested have shown significant gaps in accuracy when testing for disease-causing bacteria including Salmonella and Campylobacter. On average, participating labs had a false negative result rate (meaning bacteria was present when the test showed no pathogens) of 4.9 for Salmonella and 9.1 for Campylobacter. False positives (indicating bacteria was present when it was not) occurred at a rate of 3.9 percent for Salmonella.

Christopher Snabes, food technical specialist with the American Proficiency Institute, explains that it is unrealistic to expect that all labs would be error-free. No testing method and no testing facility is foolproof, and the largest variable is the human factor. According to Snabes, a number of errors can produce false negatives or false positives. For one thing, the lab technician can test for the wrong bacteria, or confuse samples and their target bacteria. A sample infected with Campylobacter will appear clean if only tested for Salmonella. Sometimes a recently emerged strain of bacteria has not yet been included in a lab’s pathogen database, or a technician makes a mathematical or transcription error. In proficiency testing, these mistakes result in a black mark, but in real life, the consequences can be deadly if a disease-causing pathogen finds its way into the food supply and sickens consumers.

Beyond human error, equipment problems can cause wrong incorrect results. Instrument failure can skew test results. Also, the reliability of test kits varies from manufacturer to manufacturer, and faulty kits produce incorrect results. Garbage in, garbage out.

Conventional Testing Methods

There are three main detection paradigms used to test for Salmonella and Campylobacter: Culture, ELISA, and PCR.

Culture. A pathogen test in which a sample of the food in question is placed on traditional selective culture media to allow the target microorganism to grow while simultaneously preventing the growth of other organisms. Culturing is the oldest method of pathogen detection as well as the gold standard because of its clear, visible endpoint. However, cultures are time consuming, taking several days to produce results depending on the target organism, which can be prohibitive when dealing with perishable food with a short shelf life. It also requires trained laboratory staff following Good Laboratory Procedures and, even when technicians take the utmost care, cultures are vulnerable to the interference of background flora.

ELISA. Enzyme-Linked ImmunoSorbant Assay determines if a pathogen is present by detecting the presence of an antibody that has linked to it. An enriched sample solution is placed in a 96-well plate coated with a protein that will bind to an antibody to the target microbe. The solution is removed and a second antibody, linked to an enzyme, is added that will bind to the first antibody, making an antibody-antigen-antibody sandwich that will cling to the side of the well. The solution is washed away and a substrate is added to the well that will cause the colorless antibodies to become colored products, thus signaling the presence of the target bacteria. A detraction from ELISA is its cost. A different test kit is needed for each unique strain of bacteria, and this can impact the reliability of results. Companies will do a cost-benefit analysis and test for usually two or three strains that are the most likely to appear in the products, potentially overlooking a harmful microorganism.

PCR. Polymerase chain reaction is arguably the most sensitive of the three methods because it looks for the actual DNA of Salmonella or Campylobacter strains. A sample is heated in a thermocycling instrument, splitting the double-helix DNA into a single strand. An enzyme called “Taq polymerase” is added which builds two new strands of DNA using the originals as templates, and then proceeds to amplify the DNA exponentially, creating a large enough sample of DNA for pathogen detection.

False negatives allow dangerous pathogens to be released into commerce, sickening consumers.

What’s New?

One example of a platform that claims to lower the risk of technician mistakes is the Molecular Detection Assay (MDA). Launched by 3M in 2011, MDA uses BART (Bioluminescent Assay in Real Time) technology to recognize distinct sections of a bacteria’s genome. In an email, 3M’s food safety division explained that MDA involves two processes: Isothermal DNA amplification, meaning it is done without a thermocycling instrument, and bioluminescence which uses luciferase, the enzyme that causes fireflies’ abdomens to light up. An enzymatic process in the enriched sample produces ATP which reacts to luciferase, causing the target pathogen DNA to glow. MDA reduces the risk of human error by requiring only a single instrument and preparation protocol across most assays. The technician does not have to match the protocol to the pathogen, thereby lowering the opportunity for confusion. Additionally, MDA uses color-coded assay tubes to differentiate pathogen assays to help shrink the margin of error.

Safeguarding against instrument failure is another way to improve testing accuracy. MDA does not require calibration and features an automatic diagnostic program that runs on startup.

Accuracy is paramount in pathogen testing, but speed is an important consideration as well. If a supplier has perishable food sitting in a climate-controlled warehouse, the shorter the time to results the better, and 3M claims that MDA delivers molecular level accuracy in real time. The process still requires enrichment time of anywhere between 18 to 24 hours depending on the target pathogen. But once the enriched sample is placed in the instrument, a presumptive positive can be seen in as little as 15 minutes, and a negative result takes 75 minutes.

Life Technologies, providers of a PCR test, is currently developing a technology that improves upon the conventional PCR platform. The company believes its new assay will increase accuracy and shorten time to results. The confirmation test, run after the presumptive positive, adds time to pathogen testing. In the interests of accuracy and consumer safety, it is a good idea to confirm negative results as well. However, some food processing companies skip the confirmation assay if the initial results turn up negative, although this is a risk because a false negative can allow a potentially deadly pathogen to slip into commerce. According to Nir Nimrodi, director of Life Technologies’ food safety division, the technology now under development will allow the confirmation assay to run simultaneously with the initial test, using the same sample, saving time and improving accuracy for testing laboratories.

Both false negatives and false positives of Salmonella and Campylobacter have an impact: False positives have an economic impact and false negatives lead to a health impact and potentially, an economic one as well. In the case of a false positive, pristine meat, dairy, produce, etc. will either be destroyed and thereby become a total loss, or be cooked before sale, which results in a smaller profit margin. False negatives allow dangerous pathogens to be released into commerce, sickening consumers. Many brands cannot recover from the damage that resulting recalls and lawsuits bring about–emphasizing that the importance of precision in pathogen detection cannot be overestimated.


Cowan-Lincoln is a science/technical writer based in New Jersey. She is a frequent Wiley-Blackwell contributor who has been featured in numerous publications. Reach her at mlincoln214@yahoo.com.

Proficiency Testing Process

Pathogen testing is what stands between consumers and potentially deadly outbreaks of foodborne illnesses, so accuracy in the lab is paramount. Proficiency testing is an objective means of testing lab accuracy, or as Christopher Snabes of the American ­Proficiency Institute puts it, “We…test the labs that test the food.” Unless a lab is ISO17025 certified, in which case annual proficiency testing is mandated, laboratories voluntarily submit to this analysis as part of quality control. American Proficiency Institute sends client labs two samples of food to test, each infected with different bacteria in varying concentrations. The lab tests it for pathogens using any method it choose—culture, ELISA, or PCR—and sends its findings to the Institute to check it against what was added. The Institute then posts its report on the lab’s accuracy on a secure area of its website, accessible only to the client laboratory. Clients log onto their own secure page to find out how this test rates their exactitude.

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