@Risuena - I actually looked in detail at the study you found and linked, and it did actually help me wrap my head around the whole notion of the potential for a true positive on the CFT, a false negative on a Western Blot, and the potential for this sort of situation.
The owner, trainer, and their friends are obviously frustrated with the situation and seem positive that there is an issue with USDAs protocols, and poor communication on the part of everyone involved in this situation that has turned it into the nightmare it is. That MIGHT be true. And it is perplexing that the day rate charged by Jet Pets went from $300 per day, to $1300 per day for a while, but then was reduced to $300 per day again. HOWEVER…
Isn’t it also possible that the decision by USDA to change their previous protocol, and no longer force decisions regarding euthanasia or returning a horse to the country of origin after 2 positive CFT results for glanders, and to essentially now allow an animal to linger in USDA quarantine indefinitely if they also have multiple negative Western Blot tests for the disease - perhaps that particular change in protocol is part of why this situation has turned into the nightmare it has? Is it wise to advocate for additional changes in protocol BECAUSE of this case? It seems like a case could actually be made to revert to the prior protocol… if you see what I mean. Only 2 tests. No 60 plus day stays trying to sort things out and bleeding money.
I know that some are in favor of just believing the negative Western Blots, but isn’t it possible the horse currently at LAX is actually repeatedly an example of a false negative on the Western Blot? I understand it’s very unlikely… but it seems like it is NOT impossible. I did read through that 2019 study, and it seems very clear that this actually is a risk, and that the discussion both on these forums and on the Facebook thread really isn’t acknowledging that possibility (or maybe I missed it).
So back to that study that was linked earlier. Maybe
someone else with a strong scientific background on issues with respect to these tests and interpreting the results of different studies could be kind enough to try and educate me (and anyone else who is curious and following this case) a bit more about the possibility of false negatives. Specifically, after looking at Table 2, I saw that of the 142 samples which were from actual glanders positive horses which were part of that study (confirmed via molecular detection according to the study authors 
I have no clue what molecular detection means) there was in fact 1 subject that had a positive CFT, positive ELISA-IDVet, positive ELISA-Hcpl, but a negative Western Blot.
So is it fair to call that 1 test subject an example of an actual false negative on the Western Blot? 1 out of 142?
Additionally, the study noted that they collected samples from equids in India in 2016 and 2017 during an active glanders outbreak, and these animals made up a significant part of the “glanders positive group” in this study. But some of these samples were NOT confirmed to have a Glanders infection via molecular detection (pcr). Instead… they selected study participants believed you be Glanders positive based on clinical symptoms, and intentionally selected the animals that were the MOST symptomatic (very sick) and were known to have had contact with infected animals. Of the 112 subjects which fit that description, they found 2 had a positive CFT result, positive ELISA-IDVet, positive ELISA-Hcpl, but a negative Western Blot.
Sooooo… might these 2 not ALSO be potential examples of a false negative on the Western Blot? 2 out of 112?
I realize this is only one study. But the results utilizing 4 different testing methods (some highly sensitive, some highly specific, some both) on horses that are either CERTAINLY, or ALMOST CERTAINLY infected with glanders, seem to reveal that a false negative on the Western Blot can occur about 1% of the time.
The number cited over and over with respect to false positives on the North American CFT seems to be 3%.
Given these issues… I can understand why the USDA policy is what it is, and they simply will not allow a horse that doesn’t have a CFT of 0 into the country. I can also understand why the combination of the sensitive CFT, and the specific Western Blot is a very effective way to screen for glanders. And to the point made by Pennywell Bay, why adding another highly specific test such as the ELISA-IDVet, might also help weed out a false positive on the CFT…
But those ELISA tests also seemed to indicate in this study that the Western Blot CAN, and sometimes DOES, result in a false negative.
WITH THAT SAID… One thing that I MIGHT have missed when reading the study, was any mention of whether or not they replicated some of these unusual results that point to the Western Blot giving a false negative in an infected animal. I’d imagine there might have been practical problems involved (i.e. some of the extremely symptomatic glanders infected horses who were living in India in the area with the ongoing active outbreak possibly died, and they couldn’t repeat the tests… etc…). However, I also didn’t see any indication that in cases of EITHER a suspected false positive on the CFT, or a suspected false negative on the Western Blot (or either suspected false positives or false negatives of the other 2 ELISA tests evaluated), that they went back and repeated all 4 tests on a specific subject animal, and replicated the atypical results. So maybe it was just because this was not the design of their particular study. I’m definitely noting the issue of replicating results though, because it seems like that is relevant to what is actually happening in the SPECIFIC case of “Waffles” - who is presently stuck at LAX. They have now retested this horse 4 or 5 times with the CFT, and are still getting a suspect result, but the same horse is negative for glanders on the Western Blot. Apparently… 8 negative Western Blots so far. The two other well known cases of horses stuck in quarantine with a similar issue… the Irish racehorse back in 2016 (? I think) and Sagacious HF in 2018, both got a negative CFT and negative Western Blot by the third round of testing (I believe). So there is actual proof via repeated tests on a single subject that false positive CFT results do sometimes turn negative. Eventually. But… this horse is now at 4 or 5 problematic CFTs… over a period in excess of 2 months. Are there any KNOWN cases of horses who were tested this many times and EVENTUALLY turned negative on the North American CFT? It seems like this is uncharted territory, and even though experts (supposedly even the USDA vet) SUSPECT that the horse will likely test negative with enough time… it doesn’t seem like there are public examples of this actually happening? Or am I missing something?
However… is it not also possible that there is an actual glanders infection going on with Waffles, and it is causing repeated false negatives on the Western Blot, and also caused a false negative on the European version of the CFT? Couldn’t a false negative be attributed to the fact that the Western Blot test is SLIGHLTLY less sensitive, and the European CFT, though very sensitive, uses different antigens (I think I am stating that correctly) than the North American CFT… hence WAFFLES negative on that test? I thought many many many comments ago someone linked to a study that actually indicated the North American CFT was more sensitive than the European one.
I also noticed at the very end of this 2019 study comparing efficacy of different testing methods, links to other related studies, including one from 2016… the title was, “Glanders and the risk for it’s introduction through the International Movement of Horses.” Kettle AN, et al. Equine Vet J. 2016. The abstract and summary of this study noted the following,
“The disease was eradicated from large areas of the Western world in the early 20th century, but, over the last 10–20 years, has emerged and re”emerged in areas in which it was previously unknown or had been eradicated. Although glanders was previously thought to manifest in only acute or chronic presentations, it now appears that B. mallei can produce latent infections similar to those caused by Burkholderia pseudomallei. These latent infections may or may not be detectable by current diagnostic tests.”
Additionally,
“False positives are problematic for the horse”owner and veterinary authority, whereas false negatives may allow the reintroduction of B. mallei into B. mallei”free areas. These gaps in knowledge of the epidemiology of glanders, and weaknesses in its diagnosis, coupled with the increased movement of equids, indicate that infection with B. mallei remains a major risk in the context of international movement of equids.”
Unfortunately, at that point I ran into a pay wall. So I don’t have any additional insight on what the study said about latent glanders infections. But it seems to be a very real concern. And it seems worth noting this, in the context of looking at whether or not repeated false negatives on the Western Blot are actually possible. The three false negatives from the 2019 study happened with animals who were symptomatic and very sick. I’m not a biologist… but it seems like a false negative on the Western Blot would be MORE likely in an animal with a sub clinical, latent glanders infection. Because the test is not valued for its sensitivity, but for its specificity. But false negatives actually happened in HIGHLY symptomatic animals.
One last thing I noticed… the CFT performed as part of the 2019 study examining the comparative usefulness of the CFT, Western Blot, and different ELISA methods was performed at a 1:5 dilution. In the trainer’s and owner’s comments online regarding the tests performed thus far on Waffles… they mentioned that the initial CFT result from the USDA came back at a 3. Eventually, after the horse had a fever and they ran a CBC that indicated some sort of infection was going on, and then finally got permission to have a vet come in and administer antibiotics (a shot of Exceed and SMZs I think) they said the CFT dropped to 1. HOWEVER… the trainer’s public comments in particular from December and earlier this month seem to indicate that dilutions of 1:20 and 1:10 were used when those CFTs were run. Obviously none of us know if her comments regarding dilutions are just a misunderstanding/miscommunication, and all tests were performed at a 1:5 dilution (which seems to be standard). But if her comments were accurate, and the test was performed at a dilution of 1:10 or even 1:20, and it THEN dropped from a 3 to a 1, but still did not actually drop to a zero… could the noticeable drop on the CFT simply be due to the difference in dilutions? Maybe the antibiotics did not actually impact the test results one way or another? I’m not saying that the horse therefore ISN’T cross reacting, but maybe the whole theory that he had some other sort of bug, hence the fever and the suspicious CFT that did drop (just not to 0) is actually a bit flawed?
Oh well. I’m not commenting in an effort to attack anyone. Simply to better understand why there isn’t more discussion or focus on the possibility that this horse actually IS positive for glanders?
And in all fairness to Pennywell Bay… it definitely does seem like being able to utilize an ELISA method of some type would, in fact, be VERY helpful with this case in particular in order to better understand what is going on with the horse. But unless I am missing something, that doesn’t seem to be the focus of the frustration online. People are furious with Jet Pets still. Much discussion of boycotting LAX for imports.
Anytime you have any questions, please feel free to ask.
