Julia Stewart:
Hello, this is PMA PR Director Julia Stewart, and welcome back to our “Ask Dr. Bob” audio blog series on product testing, “with PMA’s Chief Science and Technology Officer Dr. Bob Whitaker. This post is part of a series we’ve been doing on the topic of product testing. Bob, in several posts now you have indicated that sampling is actually a more significant challenge than actual testing…Tell us more.

Bob:
Julia, as we’ve talked about earlier in this series, the specificity and selectivity of pathogen tests is only half the equation – the other half is your sampling program or the method you use to collect fruit and vegetable material to be tested. From our previous posts, I think you can all see there are many challenges and benefits associated with the actual pathogen tests. Yet in many ways developing a sampling methodology that can achieve statistically significant confidence levels is more troublesome.

So, let’s start with what we know … Based on the millions of pounds of produce harvested, shipped and consumed each day by millions of people throughout the country without illness, we know that the frequency of pathogen contamination is low. We also know from data shared at the Center for Produce Safety’s Research Symposium in June 2010 that pathogens do not survive well in production environments.  Indeed, two days after purposely spraying attenuated E. coli O157:H7 on leafy greens crops, researchers could only recover it by using enrichment techniques.  (By the way, “attenuated” means the pathogen’s disease-causing gene has been deactivated so the bacteria can be used in testing without risking making anyone sick.)

So, because we face both low-frequency contamination and low pathogen survivability, it’s crucial for our sampling methods to be constructed so we can detect even sporadic, low levels of key pathogens. Further, contamination – when it does occur – it is not uniform. When contamination is found in a field, it tends to be random and isolated. That in turn can make follow-up testing a big challenge. Leafy greens growers commonly report following up on confirmed positive tests with extensive field or finished product-level sampling only to find that the  initial positive test results are seldom repeated.  The key to understanding sampling issues in produce is to understand the size of typical production lots or finished product production runs. 

Just think about a single production block of fresh spinach. Let’s say the block is 10 acres in size; that’s about a day’s harvest for a small to medium size producer.  Planted at a density of about 4 million plants to the acre, our production block has approximately 40 million plants in it.  Each spinach plant at the time of harvest has 4-6 leaves, so choosing the middle of that range, that makes the total number of leaves in our production block around 200 million. 

Today, sampling programs generally follow a “Z” pattern originally developed for pesticide residue sampling, which is a much different sampling challenge than microorganisms.  Along the “Z” pattern, the sampler chooses 15 points and collects 4 samples from each point for a total of 60 samples per block.  The size of the sample generally ranges from 25 grams to 100 grams per sample point or about 50 to 200 leaves.  That means a maximum number of 12,000 leaves are collected in any given field sample of 60 points.  These are generally mixed in a sample bag to form a composite sample.  From this composite, 50 to 200 leaves are selected to create a test sample.  So, in our block of 200 million potential leaves, our test comes down to evaluating 50 to 200 leaves. 

Another way of looking at this is a commercial spinach field has an average yield of 12,000 pounds.  In our 10-acre block, that’s 120,000 pounds of harvested product.  Using the sampling program currently employed by many in our industry, we are attempting to represent that 120,000 pounds of product by sampling about three pounds of product, and then selecting a quarter of a pound of leaves from that to actually test. 

Talk about finding a needle in a haystack!

Julia:
So why not just test more material then, Bob?

Bob:
To be sure, there many variations on the “Z”-pattern test just described.  Some are doing a “Z”-pattern test on each acre within a production block.  In the example above that would be a factor of 10 greater than just doing a single “Z” pattern on the whole 10 acres.  Others are using other patterns designed to pick up border areas as well as the center regions of a field, known as “box” or “box-X” patterns.  So while your statistics may improve by a factor of 10 or so, unfortunately you’re still in the needle in a haystack territory.  

Remember, these contamination events are random, low frequency, and isolated. You could take a thousand samples from that same production block and only minimally increase the relative amount of product tested – and just as easily still fail to sample the exact location where the potential contamination resides. 

And, by the way, the same sampling issues arise if you’re talking about testing finished product. Let’s say you are packing 60 to 100 bags per minute, which is standard for some products.  Simply removing five or 10 bags of product every hour or so and then making a composite sample gives you very similar statistics compared to in-field testing.

Julia:
So how exactly do you sample effectively to detect contamination?

Bob:
Well, with today’s technology, there’s really no satisfactory answer to that question.  There are new innovations on the horizon, such as environmental vacuums and other technologies that are being adapted from the Department of Defense, where screening vast areas for weaponized microorganisms has been a priority for several years.   However, given the state of sampling technology, it’s important to understand what sampling and testing can – and can’t – do. 

Right now, the only contamination events we can feel reasonably sure to detect would be massive breakdowns in our food safety programs.  For example, if a pesticide applicator used a grossly contaminated water source to mix pesticides and then applied them to edible portions of the product, or an animal intrusion event where the animals were indeed infected with a human pathogen. Typical sampling programs might well detect this contamination, but these types of massive breakdowns have been well managed by Good Agricultural Practice programs and so have only rarely been associated with foodborne illnesses.  In other words, if a massive breakdown occurs, there are food safety programs in place to identify these issues outside of testing, and generally producers do not harvest the crop.   

I always get back to risk-based testing. If a producer knows a risk event may have occurred like an animal intrusion, or if environmental conditions known to support pathogen survival might have been in place during production, one might increase sampling in specific locations.  Likewise, if you are producing in a field where previous potential positive samples were detected in past seasons, it may make sense to screen these fields more intensely.  It comes down to evaluating the risks associated with each production block or product run and then using the context of the physical evidence, observations, and other food safety data to better target sampling.  It means taking an active role in the sampling program and not simply putting the execution of the program on auto-pilot.

Julia:
Wow, Bob, good points to consider! Next time, we’ll explore the critical issue of raw versus finished product testing.  This is a central issue for our industry with important ramifications for supply chain logistics.

Thank you, listeners, for joining us!

Julia Stewart:
Hello, this is PMA PR Director Julia Stewart, and welcome back to PMA’s audio blog, “Ask Dr. Bob” with PMA’s Chief Science & Technology Officer Dr. Bob Whitaker. Bob and I have been talking about the challenges with product testing in produce. Bob, this time we promised to get into the role of confirmation testing. Why is it important for producers and buyers to understand the benefits and limitations of this type of testing, and what does it mean to supply chain logistics?

Bob:
In previous posts we discussed rapid tests such as PCR tests, and the benefits of those types of tests – mainly speed.  Because of the pressures our industry is under to sample, test and get results back so that loads can be harvested or shipped, speed is king as they say and these DNA-based test methods have become the standard in the industry.  But, we have also discussed some of the issues with these types of tests; mainly they are not always sensitive or accurate enough. 

We have all heard of instances where initial positive samples fail to be confirmed on follow-up testing.  It is extremely important to be sure a sample is in fact positive, because growers and processors are often making decisions not to ship or harvest thousands of dollars worth of products.  And of course, adding another round of testing costs money and time.  You all might recall that as little as a few years ago, some producers recalled product based on initial test results only to find out later that the original result might not really have been positive for a pathogen, but was simply a closely related non-pathogenic bacteria that shared many similar DNA fragments.

So, this is where “confirmation testing” enters the picture. Initial positive samples are often re-tested using another type of test.  To be clear, this isn’t just running the same test again – this is using a completely different test to determine if the first was accurate or not.  Some choose to use FDA recognized microbiological plating methods commonly referred to by many as “BAM testing” – “BAM” stands for FDA’s Bacteriological Analytical Manual.  Others use another round of PCR-based testing focusing on a more diverse set of DNA primers characteristic of a specific pathogen.  Sometimes, depending on the pathogen, there are other types of tests that can be used.  For example, if an initial test indicates E. coli O157:H7, one might opt to use an immunological-based test to determine if shigatoxin, which is characteristic of E. coli O157:H7, is present. 

Julia:
Wow, sounds like a very tricky issue. I see why you stress that anyone who is testing their products understand the benefits and limitations of each approach. 

Bob:
That’s right Julia, there are pluses and minuses for any of the approaches I just described. 

For example, using another round of PCR-based testing has the advantage of being fast.  Once you have the “positive” results from the initial testing, you can have the lab immediately run a second round of testing using a different set of primers designed to the target the pathogen.  You can have results back within another 12 to24 hours using this approach.  The logic is that the more DNA primers you test for, the more likely it is that your original positive test was correct – and, correspondingly, the less likely that the positive test was just a close cousin of the pathogen that shared some of the pathogen’s same gene sequences.  The drawback is that you have to be certain the primers you use really uniquely distinguish pathogens from non-pathogens.  The science is not entirely conclusive on this matter yet, so you have to work with your labs to understand the specificity and selectivity of the probes used in your test and the relative significance of the number of unique primers used in building the confidence that your results are accurate.  

On the other hand, BAM testing is kind of the gold standard for microbiologists.  It’s based on the ability of bacteria to grow on different media “recipes”.  Every microorganism’s DNA genetically codes for specific proteins or enzymes that allow that microorganism to use certain sugars and nitrogen sources to grow.  Different bacterial species, of course, have different DNA and therefore differ in which sugars and nitrogen sources they can grow on.  Over time, microbiologists have developed a complex set of testing media which can distinguish specific pathogens from related non-pathogenic strains.  These media recipes are well recognized and accepted by scientists and can be a very powerful tool to identify specific pathogens.  

So as a confirmation testing tool, BAM testing can be very specific and accurate.  It can distinguish between closely related species of bacteria and at the end of the confirmation test, if the sample tested positive by PCR and BAM methods, you can be reasonably confident that you have a  pathogen-positive sample and a live culture of the pathogen from the lab   However, BAM testing isn’t without issue itself. These tests take time, anywhere from 48 to 96 hours depending on how you handle samples.  It is also important to note, that there is also a degree of human discretion in reading BAM results, and bacterial colony shape and colors can be interpreted incorrectly on occasion. 

Julia:
So, confirmation testing is an important tool to verify initial positive results and give the producer and the buyer confidence that products either are or are not truly contaminated with a pathogen.  Thanks, Bob.

Next time, we’ll talk about a very complex area of concern: product sampling.  So, listeners, please join us for what is sure to be an interesting discussion.  

Remember you can email Bob at AskDrBob@pma.com. In addition to listening to these and other Ask Dr. Bob blog posts, we invite PMA members to visit our new online Food Safety Resource Center on PMA.com and check out the lab testing white paper in the Education section. Thanks!

Julia Stewart:
Hello, this is PMA PR Director Julia Stewart, and welcome back to PMA’s audio blog, “Ask Dr. Bob” with PMA’s Chief Science & Technology Officer Dr. Bob Whitaker. We’re in the middle of a continuing series on the challenges of pathogen testing in produce. In the last post we talked about how a positive is not always a positive and the importance of looking at the selectivity and sensitivity of tests.

Bob, in the last post you also said that a negative test result might not actually be a negative. That seems even more worrisome to me. I mean if you test a product and the result is negative and you ship it out only to find later that it was positive, that can be a big problem.  Can you elaborate on that?

Bob:
Sure, Julia.  There are basically three issues to look at here: enrichment, interference and sampling issues.  Let’s take enrichment first.  Enrichment is simply the method used to permit any bacteria that might be in a sample to grow and multiply so you can get enough DNA to be able to isolate it and start the process of determining if any of it comes from a pathogen.

Since pathogens are typically present only in very low levels, when present at all, this process of enrichment can be critical.  You also have to appreciate that these pathogens thrive in warm, humid, high nutrient environments — in other words our intestinal tracts and the surface of a fruit or vegetable is not necessarily the best environment for them.  This enrichment method permits these bacteria to recover, if you will, and begin growing again.  So, it’s important that conditions and times are maximized to permit pathogen growth.  This typically takes 12 to 18 hours.  Failure to use proper enrichment methods can result in pathogens going undetected even though they may have been present in the original sample.

If that wasn’t enough of a challenge, we’ve got the issue of interference. Fruits and vegetables are extremely complex in terms of their chemistry. They’re made up of thousands of chemicals, and that composition can sometimes change dramatically based on variety or growing conditions.  Some of these chemical constituents can actually interfere with pathogen detection by interfering with DNA isolation, purification or detection.  It seems like it is almost necessary to develop specific isolation protocols for every commodity to be sure the tests are selective and sensitive enough to find pathogens if they are present.

Lastly, we have the sampling issues. If your sampling program is not sufficient, it is quite possible that if there is a low level or non-uniform contamination event, like we typically see in produce, that you could simply miss including contaminated fruits or leaves in your sample.  We’ll talk about this more in a future post.  In fact, I think this is actually a more important consideration in product testing than even the tests themselves so it warrants a more extensive discussion.   But in the end, if you don’t have a dynamic sampling program, you can easily miss contamination and get a “negative” result for a field or batch of finished product that might indeed be contaminated.

Julia:
Thanks Bob.  You’ve raised some good points here.  After hearing this, I can see why you always say that food safety programs should first focus on preventing contamination from ever happening.  Detecting it after the fact is not a simple issue.  I can also see why you repeatedly emphasize how important it is for those doing testing to ask questions and fully understand what their labs are doing, and what the data may or may not mean. 

Next time, let’s dive into this issue of confirmation testing and how that can be used to verify these initial or PCR-based screening test results.  Listeners, thanks for tuning us in today and we look forward to speaking with you further on this issue of product testing.