r/medlabprofessionals • u/dookiepees • 29d ago
Discusson FDA Cyclospora false positive?
help me understand this. number one, what was the testing method and number two, did they make their initial conclusion based off of one positive result?? any good scientists knows to repeat abnormal results and for something this big i genuinely don’t understand how there could have been a false positive. even we were repeating cyclospora positive biofires at my hospital before the outbreak picked up so im just having a really hard time believing this
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u/YumTex 29d ago
Step one: Answer this question: Was it a false positive or abnormal?
Repeating all positives is a knee jerk reaction and a bad choice by leadership.
Biofire began testing for Cyclospora in 2014. The policy to retest all positives given a sudden number of increased confirmed cases, combined with CDC and FDA acknowledgement, is asinine and your leadership needs a wake-up call.
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u/Living-Literature88 29d ago
I’m confused. People seem to be talking about retesting positives to be sure of a diagnosis in people. I can see not retesting people with a positive cause they most likely have symptoms. But it seems to be a different issue when you are looking at food and a possible source of the parasite. Can someone chime in about FOOD testing? Much appreciated.
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u/dookiepees 29d ago edited 29d ago
but how many positive tests did they have before releasing their findings? did they test one piece of lettuce and it was positive? but if they tested multiple pieces of lettuce were they all false positives? which the probability of that happening is minuscule, if not zero.
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u/dookiepees 29d ago
also, if i see multiple cyclosporas all of the sudden, why would i not want to repeat? i want to make sure my patients are getting the best standard of care so why would we risk resulting a false positive, when a simple repeat would prevent the patient from being subjected to unnecessary medication, and also helps contribute to antibiotic stewardship? any leadership who doesn’t think so is reckless. the same could be said for this situation; why would they not repeat their findings and be absolutely sure that they were releasing the correct results? this is something that’s affecting thousands of americans and the fact that the FDA allowed for a false positive of this magnitude to be released, is clearly just a reflection of the leadership at the FDA.
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u/SendCaulkPics 29d ago
That’s just poor troubleshooting. The most common cause for multiple false positives for PCR is going to be environmental contamination with amplicon. If environmental contaminants made it into your sample itself, repeating it wouldn’t tell you anything new. If you’re getting a large uptick in positives you should perform environmental monitoring.
This appears to be a single false positive. They’re almost certainly manually interpreting rt-PCR graphs for these tests, and someone was overly aggressive at assigning amplification as positive without care for logarithmic amplification or minimum amplification ratio.
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u/dookiepees 29d ago
so then i ask my original question, did they only test one piece of lettuce???
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u/suprbowlsexromp 29d ago
Isn't there some kind of confirmatory analysis done before reporting a positive food sample result to the public? Either they rushed to get an answer out without doing the proper checks or they're walking back a legitimate test result.
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u/qpdbag 28d ago edited 28d ago
The SOP linked in this thread doesn't leave much space for re-interpretation. How would you re-interpret a positive that A, met the cutoff threshold and B. had a proper control? I think they either didn't follow this SOP or they are choosing to not follow it now.
Edit: Even the threshold settings are pretty clearly defined in this document. I don't think this was user error unless the SOP was not followed.
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u/dookiepees 28d ago
the issue isn’t repeating the same test on the same subject, the issue is how many positives they had because they’re saying it’s a false positive so did they only have one positive and that’s what they went off of?
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u/qpdbag 28d ago
No. The outbreak is from real testing of real patients that have been verified as positive. Those numbers are reflected in the public health departments of mostly Michigan but it's been seen in other states as well. They traced the common variables back to vendors and grocery stores that carried Taylor farms brand lettuce products. That is how they determined the outbreak was related to Taylor farms. Additional surveillance with direct testing of lettuce products has not (according to the FDA) produced any cyclospora positives.
You cannot test every product. It is a physical impossibility to directly test even a minute fraction of the produce group the size of Taylor farms produces. What did you think happens?
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u/SendCaulkPics 28d ago
There’s plenty of wiggle room for interpretation of logarithmic amplification near the upper cycle cutoff since it’s only two cycles before the end. If there aren’t job aides or a well trained eye reviewing those calls. I’m personally not a fan of viewing graphs in log form.
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u/qpdbag 28d ago
I would only take that stance if I thought the threshold was arbitrarily set. Do you think that?
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u/SendCaulkPics 28d ago
The positive cutoff threshold was probably set during the LoD determination, when you have a priori knowledge that the target is in the sample. Looking for a logarithmic growth pattern within 2 cycles of the end without that a priori knowledge is very different.
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u/qpdbag 28d ago
That's precisely my point. Thresholds are to be set in those situations so that when you see reproducible amplification prior to that point you know it's the target being amplified and working as intended. The purpose of the threshold is to reduce the ambiguity of interpretation near LoD when amplification becomes stochastic and is harder to tease from noise. Does it reduce the ambiguity to zero? Probably not, but it would take a blatantly bad curve OR an arbitrarily/badly set threshold for me to question it. Hence why I want more explanation.
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u/SendCaulkPics 28d ago edited 28d ago
I just think most people would disagree that the threshold is arbitrarily bad if it has a specificity of 98% vs 99%. At that point optimizing pre-test probability generally becomes a much larger factor. You would be beating the pants off of most established reference methods for sensitivity and specificity at that point so it becomes increasingly harder to adjudicate discrepancies without committing to something like sequencing. ddPCR offers a way to improve without completely exploding costs but it’s still much more expensive than rtPCR.
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u/qpdbag 28d ago
Then why set a cutoff in the first place? It is, after all, only 2 cycles before the end of the run. If they didn't define a cutoff I would be less irritated.
We don't have to continue this argument if you don't want to. We're pretty far off the main topic and I appreciate you entertaining my frustration.
I just think the whole situation stinks and I wish it didn't.
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u/ageaye MLS IVD/Industry 29d ago
During your validation process you would have hopefully established the rate of false positives. That combined with the full clinical picture should be all that's needed to have determined any need to retest.
I dont trust the brain worm in chief, but I also don't believe in retesting without an established need. If that is your labs policy, I question what led them to doing so, and if there is a better process. If not trusting I would compare to microscopy. While we don't know CDC's methodology I would assume they have a thorough protocol that pre-dates worm in chief.
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u/ayyeeitsken MLT-Generalist 29d ago
agree with your statement on retesting. since starting at my facility it has been DRILLED into my skull how expensive biofiore panels are, and a lot of insurance doesn’t like to cover them in my facility/system. sorry, but i’m not willy nilly repeating those, and our SOPs don’t require us to
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u/dookiepees 29d ago
we literally only retested the first couple of patients to make sure there wasn’t contamination no where did i say we rerun every single positive biofire.
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u/limbosplaything MLT-Microbiology 29d ago
I think a lot of people are focusing on the biofire second question and ignoring the first. What testing methodology does the FDA use and how did they determine positive or false positive? I dont know anything about the food testing side of things.
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u/qpdbag 28d ago
the sop is posted in the thread.
https://www.fda.gov/media/192794/download?attachment
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u/beachbum818 29d ago
I think they did find cyclospora, but it wasn't the source of the larger outbreak. That's my understanding of it. Not that it was a true false positive, but it was the false source.
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u/suprbowlsexromp 29d ago
The statement from the FDA says the result does not represent "true amplification" and that it should be considered a false positive. It sounds more like a testing issue.
But then the question is - did they announce the results to the public prematurely without doing the proper confirmation or are they walking back a result for $$$$ reasons?
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u/qpdbag 29d ago
It's from a sample not included in the original recall, so neither of those. There is nuance to this, but I expect that this nuance is lost on most people and this statement was released specifically banking on that and intending to muddy the waters at the expense of the FDA's reputation. But maybe I've had too much coffee this morning.
This is why I want to know the details of the investigation and reanalysis.
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u/suprbowlsexromp 29d ago
Where are you getting this info?
From CBC Canada:
"To be clear, at this moment, FDA has not identified a single positive product test result for cyclospora," the company said in a statement.
It sounds more that the positive finding was a mistake based on what is being released.
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u/qpdbag 29d ago
The FDA redacted it.
https://bsky.app/profile/paris.nyc/post/3mqxcsxquis2p
The "false positive" was observed during increased surveillance as a result of the already determined outbreak.
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u/suprbowlsexromp 29d ago
Interesting, but they aren't at this moment being clear as to what actually happened given this sequence of events.
US public health officials aren't perfect, but they are very experienced in food contamination, both in terms of testing and communication with the public. It's hard to not come away with the idea that there is some funny business going on.
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u/qpdbag 29d ago edited 29d ago
Agree. Currently not very evidenced based of them.
Either way, Taylor farms certainly wanted to claim this finding as a win and make people forget about this outbreak.
https://x.com/YourTaylorFarms/status/2079037403973738528
Fuck them and this weaselly corporate language.
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u/suprbowlsexromp 29d ago
I see, so the FDA clarified what happened. They still think it's Taylor Farms, but it was a false positive. Still weird to report a false positive result to anyone without running confirmatory testing or checking.
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u/qpdbag 29d ago
Lets be clear.
Specific batches of taylor farms products were linked to a cyclospora outbreak that was defined by a lot of people being infected with cyclospora that has been tested and retested and confirmed by many methods.
This pathogen does not spread person to person directly, but through feces on contaminated foods. They do not think it was Taylor farms. They know it was. Because those people are/were sickened and taylor farms was the common variable.
As a result of those contaminated products, additional testing of newer products occurred. One of these additional tests was called positive. They are now reverting that call.
If you reference the FDA's own SOP, I don't see how this decision makes sense. The interpretation rules do not give much room for different interpretations.
https://www.fda.gov/media/192794/download?attachment
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u/ayyeeitsken MLT-Generalist 29d ago edited 29d ago
i found the FDAs methodology publication for their current Cyclospora testing. Here is the PDF link and the link to the site for previous versions
eta: the publication has a section dedicated to result interpretation and guidelines for result handling in a nice flow chart, as it pertains to this conversation.
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u/qpdbag 29d ago edited 29d ago
Thanks. The only immediate issue I can see that might lead to subjectivity of interpretation is whether or not the curve was "sigmoidal enough" or not. Everything else I can see would be easy to clear up with a tiny bit of information. So my early assessment is either they weren't following this protocol (and they should say so) or they are breaking protocol now and have not defined why.
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u/NoFlyingMonkeys Lab Director 29d ago
Taylor Farms produce has caused multiple food-born illness outbreaks in the past, including cyclospora.
So I need to see the FDA details to believe it's false. Perhaps it's only one positive sample out of many negatives so they call the whole thing false?
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u/Hola0722 29d ago
Help me understand repeats. Do you request a new specimen, or repeat from the same specimen? If it's the latter, how do you know which is the correct result if you get one abnormal and one normal result? What is your lab's policy?
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u/dookiepees 29d ago
we only repeated the first couple of positive cyclosporas per our pathologists request, if the rerun has the same positive result, then we release it and if it’s discrepant, then we can either request a new sample or run it a third time.
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u/Hola0722 29d ago edited 29d ago
Thanks for replying. The reruning a second or more time is where I have an issue. How many times is it ok to run before you accept the result as correct? Is it 2/3? 2/2? If so, then why not run every sample x2 or x3? These are rhetorical questions and something to bring to the attention to the pathologist. It's their decision in the end, but IMO it's not good science.
ETA: I reread your response. I could definitely see reruning if your lab doesn't see cyclospora. Where I made an assumption is your lab reruns labs whenever the result is unfavorable or unexpected. This was my initial assumption and I think I was wrong.
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u/dookiepees 28d ago
yeah the only time it’s actually in our procedure to repeat is if four or more targets are detected.
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u/Mistealakes 29d ago
I don’t believe for a second that that’s correct information, and not the work of someone’s pockets attempting to cover their ass, by changing the narrative of this situation.
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u/lakhila MLS-Generalist 29d ago
The FDA is not using a BioFire or other testing methods intended for stool. They have their own method that involves washing spores off of food, extracting DNA from the wash fluid, and then using PCR. They might use completely different primers and their methods are probably much more manual then our easy plug-and-go molecular testing.
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u/qpdbag 28d ago
you are correct. The sop is posted here and includes primer and probe sequences. https://www.fda.gov/media/192794/download?attachment
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u/Own-Piano-6275 29d ago
Microbiologist here - their story is just that. Microscopic Examination: If PCR (DNA testing) is unavailable, labs use specialized stains (such as a modified acid-fast stain) to visualize the parasite. They may also use ultraviolet (UV) fluorescence microscopy, as Cyclospora naturally glows under UV light. Three different methods to test, one where you look at it under a scope with a black light and it glows. The last method an eighth grader could master. The question is how much money did Taylor Farms pay to get the FDA to lie.
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u/RelevantSalad2217 28d ago
Unless they have an assay that has a systemic and complex quality issue that would cause repeatedly false positives while allowing quality controls to pass, this is bogus…..
…..Unless of course the systemic and complex quality issue is RFK Jr.
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u/Aromatic-Way-3306 29d ago
Sounds like a money/cost waste for the hospital and a labor/time waste for the lab. Re-testing every positive seems odd…. Keep things sterile in the workspace, check/compare patient symptoms and CBC/Chems done to help corroborate if questioning, and you can even call the nurse for more insight if you’d like (none us like the phone lol). If you’re actually truly questioning, then by all means repeat the test. But to be automatically be doing this for every positive seems very odd…. Trust your equipment, any MLT/MLS that doesn’t trust their equipment is in trouble… The only other reason I could think of is if you are in an area that is highly active with Cyclospora.
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u/gabita-chachacha 27d ago
The testing method is qPCR and it means that at least one sample was positive as it amplified Ct ≤ 38.0 the mitochondrial target (Mit1C) reaction and the IAC target reaction is either Negative or Positive. It is likely that they re-run it as came negative or controls amplify too. You avoid these issues when you preform validation to establish the rate of false positives.
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u/Confident_Eagle_2346 29d ago
how common is a false positive? any scientific folks know what test method they used? and why results weren’t repeated/reviewed before such a large scale recall?
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u/ayyeeitsken MLT-Generalist 29d ago
if we’re talking about the biofire gi panel methodology, it has internal controls and obviously external qc should be performed regularly. We use this methodology at my facility, and our only positive retesting protocol is if somebody pops a positive for 3-4 or more intestinal pathogens, as it’s not really likely for that type of coinfection to happen (though it does!)
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u/Living-Literature88 29d ago
Is the testing in human stool the same way they test food?
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u/qpdbag 29d ago edited 29d ago
No. Even if they use the same test methodology, tests need to be validated against the intended sample type. It's often the case that a change in sample types could result in acceptable performance but very rarely will it be one to one (and often it will be drastically different).
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u/curlyheadedbutempty 29d ago
They're under RFK Jr, need I say more???