# Disturbing News on PCR Testing for DB



## Web Wheeler (May 9, 2010)

I found this while revewing the comments on the proposed USF&WS listing:



> Before USFWS can require this testing, serious issues must be addressed regarding PCR. 1) Currently there is ONE commercial lab offering PCR testing. Cost is $20-30/sample and although samples can be batched (5 swabs/batch) the cost to importers is prohibitive. 2) Housing would need to be provided to maintain amphibians while awaiting test results. Facilities for this do not currently exist, cost is prohibitive and this type of housing would seriously increase the spread pathogens/parasites. 3) PCR testing is NOT accurate. I have conducted BD research since 2003 (all published). During one doctoral study I had 20 infected animals that I followed via histology. I am an expert at histological diagnosis of BD. I collected skin slough, prepared slides and viewed them daily. At the end, I took swab samples and sent them in for testing. ALL came back negative. I tried to work with the lab, troubleshooting swabs, ETOH, etc., yet after three rounds of PCR testing all with the same result, it was clear that something was very wrong with PCR. As a disease ecologist, I will say that the academic community?s labeling of PCR as the gold standard for BD testing was highly premature. Gold standards take decades of testing to ?proof? them. BD PCR testing has not been through the type of rigorous testing that true gold standards have. I have first-hand knowledge that PCR is NOT accurate for BD testing. I strongly advise USFWS that these issues must be resolved first. We need multiple labs that offer the test at low cost, to handle the sheer volume of animals that are imported. I also suggest an easier alternative to testing: assume infection and require prophylactic treatment instead. I and my colleagues recently found a treatment that is cheap and 100% effective. Cheaper than testing, but facilities would still be needed and personnel to conduct the treatments and care for animals. The in-press manuscript is attached. Please feel free to contact me. Dr. G.E. Padgett-Flohr


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## Ed (Sep 19, 2004)

Web Wheeler said:


> I found this while revewing the comments on the proposed USF&WS listing:


There are multiple labs doing it.. Chytrid Fungus « Amphibian Ark

The success rate of swabbing has been demonstrated under critical conditions see http://www.int-res.com/articles/feature/d073p175.pdf 

The only time histopathology was shown to be more effective is if the frog has been infected for 4 days or less. 


Ed


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## Web Wheeler (May 9, 2010)

Ed said:


> There are multiple labs doing it.. Chytrid Fungus « Amphibian Ark
> 
> The success rate of swabbing has been demonstrated under critical conditions see http://www.int-res.com/articles/feature/d073p175.pdf
> 
> ...


I haven't read the whole document, but I couldn't help noticing that Kriger and Hero are two of the authors of this study.


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## Azurel (Aug 5, 2010)

Ed said:


> There are multiple labs doing it.. Chytrid Fungus « Amphibian Ark
> 
> The success rate of swabbing has been demonstrated under critical conditions see http://www.int-res.com/articles/feature/d073p175.pdf
> 
> ...


In the first quote the guys say's "commercial" testing.....Which is very differant then research testing since most research labs are closed off from the public.....There maybe 4 labs here that do it but if you as a commercial enterprise or privet citizen only have a single lab as an option the back log would be sever and I am sure you and me as privet hobbyist would be given back burner status to commercial business's.


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## Ed (Sep 19, 2004)

Web Wheeler said:


> I haven't read the whole document, but I couldn't help noticing that Kriger and Hero are two of the authors of this study.



Is there any particular reason you are attempting to discredit the data in the paper based on the authors? Can you back up a claim that the data and results in the paper are incorrect or the results of poor science?? Particularly in light of the publication in a heavily peer reviewed journal where any inconsistencies would have gotten flagged.... 

Is this one better? It doesn't include Krieger and/or Hero.. http://www-lbtest.jcu.edu.au/school/phtm/PHTM/frogs/papers/boyle-2004.pdf

There is a lot of literature out there that indicates that histopathology can also have a lot of problems when the frogs are lightly infected as the biopsy sample may come from either a very lightly infected area or an uninfected area. 

Part of the premise with your posting that this is a problem for the TWI PCR tests is that you do not know the method being used to run the PCR. Some of the earliest were not as accurate but in the last few years we have seen a significant improvement in the ability for it to detect as little as one zoospore in the sample... 


Ed


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## Ed (Sep 19, 2004)

Azurel said:


> In the first quote the guys say's "commercial" testing.....Which is very differant then research testing since most research labs are closed off from the public.....There maybe 4 labs here that do it but if you as a commercial enterprise or privet citizen only have a single lab as an option the back log would be sever and I am sure you and me as privet hobbyist would be given back burner status to commercial business's.


As demand has picked up for the testing the turn around has improved. What in the past was a delay was waiting for enough samples to justify the costs of running the tests. Now that more submissions are getting sent into the labs, a quicker turn around is occuring. 

Ed


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## Azurel (Aug 5, 2010)

Ed said:


> As demand has picked up for the testing the turn around has improved. What in the past was a delay was waiting for enough samples to justify the costs of running the tests. Now that more submissions are getting sent into the labs, a quicker turn around is occuring.
> 
> Ed


That's good to know.....


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## johnc (Oct 9, 2009)

Ed said:


> Particularly in light of the publication in a heavily peer reviewed journal where any inconsistencies would have gotten flagged....


I am in no way referring to the specific journal article mentioned earlier, but I do want to point out that I admire your faith in our ability to review journal articles and catch everything, Ed. The peer review process often consists of throwing crap at a dart board - eventually some of it will stick, i.e. you may not be able to get a dodgy piece of info past most reviewers, but there are plenty of instances of where it does get by the panel of reviewers. And it may stick the first time - reviewers are not perfect, even in the top flight journals, and I say this speaking as an author and as a reviewer myself. 

Plus, scientists will write a paper that won't be accepted by journal X, for example the data may not be wonderful or something, but journal Y, which may not be held in the same esteem, may be willing to let that not-so-wonderful data in.

Just to reiterate, I am not referring to any paper in particular.


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## Ed (Sep 19, 2004)

johnc said:


> I am in no way referring to the specific journal article mentioned earlier, but I do want to point out that I admire your faith in our ability to review journal articles and catch everything, Ed. The peer review process often consists of throwing crap at a dart board - eventually some of it will stick, i.e. you may not be able to get a dodgy piece of info past most reviewers, but there are plenty of instances of where it does get by the panel of reviewers. And it may stick the first time - reviewers are not perfect, even in the top flight journals, and I say this speaking as an author and as a reviewer myself.
> 
> Plus, scientists will write a paper that won't be accepted by journal X, for example the data may not be wonderful or something, but journal Y, which may not be held in the same esteem, may be willing to let that not-so-wonderful data in.
> 
> Just to reiterate, I am not referring to any paper in particular.



Hi John,

I have faith that if it gets through the first process, a refutation will appear if the data etc is dodgy (often quite quickly in high profile areas..). However when there is a body of publications that agrees with the data both pre and post publication, I have a much stronger faith in that section of main stream peer reviewd data is correct. 


What I am questioning is the validity of challenging a paper on the sole basis of who one of the authors is when it is in a peer reviewed main stream journal as a method of attempting to discredit a citation without any proof that the data/results are incorrect or dodgy. 

In addition, when we are comparing peer review publications against selectively chosen "anonymous" quotation(s) (anonymous as no author was attributed here) posted in response to a rule proposal as "proof" of a failure of an ongoing test.. the weight is going to have to be allocated to the body of evidence that supports the published peer reviewed information... 

Ed


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## Web Wheeler (May 9, 2010)

Ed said:


> I have faith that if it gets through the first process, a refutation will appear if the data etc is dodgy (often quite quickly in high profile areas..). However when there is a body of publications that agrees with the data both pre and post publication, I have a much stronger faith in that section of main stream peer reviewd data is correct.


Let me give you some facts, Ed. Take, for example, the journal EcoHealth:



> EcoHealth is an international, peer-reviewed journal focused on the integration of knowledge at the interface between ecological and health sciences.
> 
> Source: EcoHealth. Global Change and Human Health


Add to the above, this quote which was published in EcoHealth:



> Preventing disease spread into naive amphibian populations can only be accomplished by removing the source of the problem: the translocation of infected amphibians. *Unlike zoos and laboratories, whose conservation services render the translocation of amphibians an occasional necessity, the amphibian pet and bait trades are for the large part disposable, that is, they are unnecessary, serving little benefit to society.* Their nearly complete dismantling would benefit amphibian populations, not only by eliminating a primary source of disease transfer, but also by simultaneously reducing the over-harvesting of wild amphibian populations, which is largely unregulated in many parts of the world (Li and Wilcove, 2005; Schlaepfer et al., 2005). At a minimum, these trades should be restricted to local sales of captive-bred individuals.
> 
> Emphasis in *bold* is mine (W.W.).
> 
> Source: http://www.savethefrogs.com/kerry-kriger/pdfs/Kriger-2009-Future-Panzootics.pdf


Then you tell me how a sweeping, highly inflammatory, unsubstantiated person opinion, such as the one I put in *bold* above, could survive a peer review process such as the one you have described?

Add to that the very recent posts here on DB by Dr. Kriger, who I am quoting:



> The paper you cite for which I am the lead author was written under the stated affiliation of "Centre for Innovative Conservation Strategies, School of Environment, Griffith University, Gold Coast, Australia", so you need not extrapolate those views to the views of SAVE THE FROGS!, which has a 7 person Board of Directors, a 30+ person Advisory Committee and many members in and out of the pet frog world.


and contrast this statement with the following statements which Dr. Kriger gave to the California Fish & Game Commission on April 8, 2010:



> Though assertions have been put forth that this ban improperly targets Asian communities due to the lack of a concurrent regulation of non-native frogs and turtles for use as pets, two wrongs do not make a right: a lack of proper regulation of the pet industry does not justify a failure to act intelligently with the food industry. Indeed *I also urge the Department of Fish & Game to consider similar regulations on the import of non-native frogs and turtles for use as pets.*
> 
> Emphasis in bold is mine (W.W.).
> 
> Source: http://www.savethefrogs.com/frogblo...ortation-of-non-native-frogs-for-use-as-food/


and this:



> They defended the frogs’ legal rights when the government failed to do so, and they fought to create necessary laws when none existed.
> 
> Source: The Story of SAVE THE FROGS!


And then tell me that Dr. Kriger doesn't have an agenda. To be sure, I'm not questioning the paper itself, only the motives of Dr. Kriger.



Ed said:


> What I am questioning is the validity of challenging a paper on the sole basis of who one of the authors is when it is in a peer reviewed main stream journal as a method of attempting to discredit a citation without any proof that the data/results are incorrect or dodgy.


As I posted, I haven't read the entire document. 



Ed said:


> In addition, when we are comparing peer review publications against selectively chosen "anonymous" quotation(s) (anonymous as no author was attributed here) posted in response to a rule proposal as "proof" of a failure of an ongoing test.. the weight is going to have to be allocated to the body of evidence that supports the published peer reviewed information...


Anonymous? Please feel free to contact me. Dr. G.E. Padgett-Flohr.


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## Ed (Sep 19, 2004)

was the quoted anonymous citation from the USF&W website yours? If so where did you indicate that was your quote? 

Please indicate where in the reference I cited; anything in that paper shows any of the bias you are attributing to that author? 

The citation you referenced was published in the Forum of the journal.. Which is the OP-Ed section where personal opinions are allowed... did you bother to verify whether it was an opinion piece before you posted it here? 

As for anonymous, as I noted above, I was referring to the unattributed quotes with which you were attempting to discredit the study....


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## Web Wheeler (May 9, 2010)

Ed said:


> was the quoted anonymous citation from the USF&W website yours? If so where did you indicate that was your quote?


No, of course not. I'm not Dr. G.E. Padgett-Flohr, the author of the quote I cited, which is included in the quote. Did you miss that?



Ed said:


> Please indicate where in the reference I cited; anything in that paper shows any of the bias you are attributing to that author?


If I find any indication of bias, I will certainly let you know.



Ed said:


> The citation you referenced was published in the Forum of the journal.. Which is the OP-Ed section where personal opinions are allowed... did you bother to verify whether it was an opinion piece before you posted it here


OK, I thought all research papers published in peer reviewed journals were peer reviewed. If I am mistaken, then thanks for the correction.



Ed said:


> As for anonymous, as I noted above, I was referring to the unattributed quotes with which you were attempting to discredit the study....


Unattributed? Dr. G.E. Padgett-Flohr was the author of what I posted and whose name appears at the bottom of the post. Again, did you miss that?


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## Ed (Sep 19, 2004)

It would have been hard to miss if you had included a link in the first post showing the source particularly since there is a supposed manuscript.. 

I did miss the end at the end of the first post but that also did not tell me anything about the author.. Does the manuscript show any reason why he had problems with Pices or was that the abstract to which you linked or was that potentially due to experimental error on the part of the person collecting the swabs? Was it before they got the system to the point where a single zoospore could be detected? 

On this note, I should point out that if you are using his comments in an attempt to discredit swabbing, then you are taking his point of view that only histopathology is sufficient (or both may be required) to detect Bd (which is not supported in the general body of the literature due to errors in collecting the samples). If that is the case then the only way to get an animal declared negative would be to require extensive biopsying which will require a vet.. and a vet to send in the sample. If a system is going to be implemented, using a swab can at least be done by a person at home.. I would be very careful on what you wish for... 

For those who really want to see a lot of the literature out there on chytrid I recommend Articles on Batrachochytrium dendrobatidis: the amphibian chytrid as a lot of the papers are free for public domain...


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## Roadrunner (Mar 6, 2004)

So if these guys can get it wrong, how are frog hobbyists going to fare? Wouldn`t you be able to fake tests by swabbing frogs backs instead of the inner thigh area? How is anyone going to be sure that the frogs were even swabbed right or at all by large lot importers or hobbyists swabbing for the first time in the privacy of their own home? Is this going to be a mandatory honor system test?

They don`t even check everyones bait now for VHS and these people are fishing out in public(and this disease affects sports fish, which get priority over frogs). Do we really think they`ll end up being able to enforce anything that goes on being shipped(And not always known by the carriers) in unmarked boxes and aren`t displayed outside peoples homes?

http://www.jcu.edu.au/school/phtm/PHTM/frogs/papers/kriger-2006d.pdf

It should be noted that the swab-PCR technique is
not without error, as exhibited by: (1) the presence of
equivocal results, and (2) the failure to detect Batrachochytrium
dendrobatidis on the single frog that
tested positive by histology. Equivocal results can conceivably
arise from low-level contamination during
laboratory analysis, in which 1 or 2, but not all 3 wells
of the triplicate are exposed to B. dendrobatidis from
an outside source (e.g. airborne zoospores or technician
error). They can also be obtained if the actual
number of zoospores present on the sample is very low
(1 to a few). This is due to the fact that the sample must
be diluted prior to PCR analysis, and only a small portion
of the original sample is analyzed in each well of
the triplicate. Thus it is possible that chytrid DNA may
not end up in all 3 wells of the triplicate. The number of
equivocal results we obtained through the PCR assay
(n = 8) was roughly equal to the number of suspicious
results we obtained through histology (n = 6), and is
therefore no more of an issue than it was with the older
techniques. However, equivocal results can be reduced
in future studies by re-analysis of the supernatant
that remains from the original DNA extraction
process. The failure of the swab-PCR technique to
detect B. dendrobatidis on the frog deemed positive by
histology may be due to the swab not having been run
over any part of the frog’s body that was infected. The
swab technique we used consisted of only 5 strokes of
the swab over the frog’s body, and therefore was not
comprehensive. Increasing the number of strokes
should reduce the likelihood of a false-negative.
Even with these potential shortfalls, the swab-PCR
technique remains an excellent tool for the diagnosis of
chytridiomycosis infection in wild amphibians, and
should greatly improve our ability to acquire the data
necessary to more thoroughly understand chytridiomycosis
and to conserve remaining amphibian populations.


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## Web Wheeler (May 9, 2010)

Ed,

Please reflect on the following:

1. "I found this while revewing the comments on the proposed USF&WS listing:" In my opinion, this is hardly an attack.

2. I provided a link to who Dr. G.E. Padgett-Flohr is a couple of posts back, via one of her research papers, in response to your "anonymous" claim. You must have missed that as well as Gretchen E. Padgett-Flohr is most likely not a "he".

3. If you had asked earlier, I would have been happy to post the additional manuscript info, but you could easily find that for yourself on the Regulations.gov website by searching for Docket No. FWS-R9-FHC-2009-0093, as I posted on this thread, which also included the same citation that I posted here on this thread.

4. I'm sorry to be so defensive here, but I feel as though I'm the one being attacked when all I did was to provide information for the benefit of DB members. Note the order: I posted, you commented, I defended...


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## Web Wheeler (May 9, 2010)

Ed said:


> The citation you referenced was published in the Forum of the journal.. Which is the OP-Ed section where personal opinions are allowed... did you bother to verify whether it was an opinion piece before you posted it here?


Please pardon my ignorance, but would this indicate a peer review?



> ACKNOWLEDGMENTS
> We thank Jeremy Thompson, Monique Van Sluys, and two anonymous reviewers for comments that greatly improved the quality of the final manuscript. Financial support for this work was graciously provided by the Eppley Foundation for Research.


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## Roadrunner (Mar 6, 2004)

Yes, but it doesn`t answer whether there is more than one lab open to public testing. The others are zoos and universities, not a private lab.


Azurel said:


> That's good to know.....


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## Azurel (Aug 5, 2010)

frogfarm said:


> Yes, but it doesn`t answer whether there is more than one lab open to public testing. The others are zoos and universities, not a private lab.[/QUOTE
> 
> True....I am sure the zoos and Unis won't be opening themselves up anytime soon either, with or with out this regulation....


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## Roadrunner (Mar 6, 2004)

What is quicker? 1000+ frogs being housed at the border awaiting test results can die quick if not fed and housed correctly. That system would add a bit more cost than even the pcr tests. And this is what Dr. Kriger and others want to happen. If this passes they then push "the next panzootic which we don`t yet have tests for" to ban all movement of amphibians.



Ed said:


> As demand has picked up for the testing the turn around has improved. What in the past was a delay was waiting for enough samples to justify the costs of running the tests. Now that more submissions are getting sent into the labs, a quicker turn around is occuring.
> 
> Ed


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## frogdoc (Feb 12, 2011)

In response to the numerous comments. I am an expert in BD and have been researching this pathogen for almost 10 years. I gave the very first talk on the pathogen and disease at the DAPTF conference in 2002; at that time few folks were studying this, and most herpetologists had never even heard of it. The reason that most folks are unaware of the problems with PCR is that PCR became a "gold standard" before it was properly vetted. Less than 10 papers on the accuracy of the test, does not make it a gold standard. Folks were very worked up about it, because histology does require a great deal of time and expertise. It took 20-30 years for TB testing to be touted as a gold standard. For a test to be a "gold standard" there has to be a test that is both sensitive (get a positive when it is positive) and specific (get a negative when it is negative). PCR for BD is sensitive, but not specific. The papers caused researchers to flock to PCR- almost no one has animals in the lab that they follow and conduct histo, as well as PCR on. I did- for four years. There is a problem and it is being disregarded. No one wants to hear about this, and other experts are quick to discount my experience. I am not alone in the experience. My current research on the pet trade, which is in prep, actually shows that animals are arriving in the US clear of BD, but are becoming infected when they get to the states. This lawsuit which precipitated this action from USFWS is based on a grand idea; but it is not workable. Where will animals be housed while testing is being done? Who will cover the cost and labor of maintenance? What lab in the world could handle the more than 5 million amphibians imported into the US? Most of the labs listed as available are out of the country-the two in the US, Pisces and San Diego Zoo would not be up to the task. 
Dr. G.E. Padgett-Flohr


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## JJuchems (Feb 16, 2004)

frogdoc said:


> Most of the labs listed as available are out of the country-the two in the US, Pisces and San Diego Zoo would not be up to the task.
> Dr. G.E. Padgett-Flohr


San Diego Zoo will not run test for the private sector. It violates the terms of their grant. I have contacted all the USA labs listed http://www.amphibianark.org/the-crisis/chytrid-fungus/. The turn around time is still 8-14 weeks.


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