Re: QPCR Uses - A Question

[Guest guest]

What methods of testing are available so I can reasonably state with

any kind of certainty to my client that the residue from ice blasted

mold remediation is "denatured?" Would the presence or absence of DNA

(through PCR) be sufficient?

Again, please ignore the several other issues involved and focus on

whether or not the mold is "denatured" by ice blasting and how that

would be demonstrated.

Carl,

Denaturing of proteins involves changing the 3-dimensional (tertiary) structure of these molecules usually by destruction of cross-linkages. I don't think dry ice works to alter molecular structure at all. It justs physically abrades/pulverizes the larger pieces into smaller pieces of the same molecular structure. Some protein molecules may be physically denatured but certainly not all.

My question to you is why would you want to demonstrate to your client that "denaturing" of allergens has been effective at all? The burden of proof should be on those claiming that the CO2 treatment somehow has rendered the "mold dust" non-allergenic. But I'm sure you've already considered this.

To answer your question, I think PCR testing could be used to show that molecules, in general, from the blasted mold were not atomized. The question about the tertiary structure and therefore the allergenicity of proteins would remain even if the DNA was intact for PCR detection. There may be some ELISA analytical methods that relate more directly to allergenicity of proteins. Again, why not just take the position that the allergens are there and let someone else try to prove otherwise?

Steve Temes

[Guest guest]

Carl, Very good question. I was talking to several persons in Nashville regarding the same issue. The culture method and direct exam are not very useful in PRV for blasting method (sand, soda, dry ice). More R & D is needed for detecting fragmented (denatured?) biomass. I would say possible measurements are DNA (universal primers/probe for fungi), ATP, enzyme (Mycometer), glucan (unreliable), allergens, etc. Blasting is effective in cleaning, but it also creates very small pieces of biomass, which may or may not be more allergenic than whole spores/cells. We need to find a new way for PRV sampling. Wei Tang QLab "Carl E. Grimes" wrote: Wei and others,Would QPCR analysis, or another technique, identify "mold" in the following situation? As I write this I'm increasingly aware of other analysis issues but I'll try to keep it fairly focused.Ice blasting was used to remove mold from OSB decking in a 2600 sq ft crawlspace. The remediator did not remove the significant residual dust that settled on the installed barrier over the soil. He/she claims removal isn't necessary because it's in the crawlspace and, besides, previous testing of such residue has always failed to identify the presence of mold. It has not previously caused him to fail PRV. The builder, who is

paying for the work under his warranty, supports the remediator.The owner of the considerably valued home is highly allergic to mold, as is one of his children. He will agree with the remediator and the builder to leave the dust as long as it is dust and not mold residue. (It should be removed anyway but that isn't the question here).The issue is the presence of triggers for allergic reactions, especially the proteins.The highly destructive nature of ice blasting may have rendered spore-based analysis unuseable. While ice blasting renders spores unidentifiable by either culturing or microscopy, does that necessarily mean the same is true for the allergy triggering proteins? Or the other components of the mold growth organism such as glucans, mycotoxins and enzymes. But I know of no studies either way.What methods of testing are available so I can reasonably state with any kind of certainty to my

client that the residue from ice blasted mold remediation is "denatured?" Would the presence or absence of DNA (through PCR) be sufficient?Again, please ignore the several other issues involved and focus on whether or not the mold is "denatured" by ice blasting and how that would be demonstrated.Carl GrimesHealthy Habitats LLC-----> Bob:> > Thanks for the info on PCR. It provided some insight and answers to > some of my questions and suspicions of PCR. Yes...Comparative > results are warranted. Moreover, the cost of PCR/QPCR tends to make > one collect, and rely on, fewer samples; and this is VERY risky when > assessing biologicals. Appreciate the input.> > -- > Geyer, PE, CIH, CSP> President> KERNTEC Industries, Inc.> Bakersfield, California> www.kerntecindustries.com> > > > On 11/26/06 7:08

AM, "Bob s" <BobBsafety-epa> wrote:> > , > > "As many of you may know, several recent scientific studies have > looked at species analysis from culture testing and found that they > are pretty much random when compared to DNA profiling of the samples > (called Quantitative PCR). Therefore culture testing for speciation > is of no value." > > Can you provide references for this statement? Since QPCR only > analyzes for some 30+ species, I have difficulty with this statement. > Further, culturable methods used in microbiological diagnosis of mold > infections in people have proven to be more than adequate from many > years. This QPCR claim is in direct conflict with medical research > methods. > > Further, at this point in time, there is no published scientific > evidence showing

that QPCR has a stronger (higher correlation > coefficient) to mold related symptoms or disease, than any of the > other test methods. > > The published research history showing a relationship between > dampness, mold and respiratory symptoms was well established in many > studies in the EU, almost 10 years ago. These studies were recently > duplicated in the US. However, this was not new information. > > There have also been studies in Germany and Canada that correlated > culturable levels with symptoms. Most other studies relate also > culturable levels to symptoms. This is why all of the mold exposure > standards in other countries are set in culturable levels. > > The only guideline on total spore concentrations is the AAAAI table. > However, note that this table drastically changed in 1992, when > AAAAI switch to Burhard from the Rot o Rod samplers. >

> There is also no study that has proven that total spore sampling is > any better at predicting mold related symptoms than culturable > sampling. The key here is the strength of the correlation. P < > 0.01 or 0.05 are equivalent in scientific power. Neither prove one > method is better than the other. > > Some people make the assumption that because hyphae or other mold > fragments can produce an allergic type reaction in a laboratory test, > that these products are a major source of the symptoms in people. > The fact is there is no data that show hyphae alone cause mold > related symptoms in people. It is extremely difficult to isolate > hyphae from the mold spores. It may be that you need 100 hyphae to > produce the same symptom as 1 mold spore. We simply do not know what > exactly is going on immunologically. > > This brings use back to QPCR. All analytic

methods have a standard > analytical error. The SAE follows this rule. culturable<total > spore<<<< QPCR. > > The problem with QPCR is if you screw up, you are easily off by a > million. With the other analysis methods, you may be off by a factor > of 10. > > I have worked in the drug industry with PCR and these analytical and > production errors are NOT uncommon. Lots of stuff gets thrown always > because of this. > > I have also seen QPCR results from clean, 1 year old wood, with no > visible mold growth, that the AIHA accredited lab reported levels of > 52,000,000. These same surfaces were negative on culturable > methods. > > The thing that really bothers me with the EPA pushing QPCR, is that > they do not do culturable and total spore parallels in ALL of there > research. > Why not? EPA goes through all the trouble

to get an expensive QPCR > research project funded, and then they don't use historical methods > as a reference to the research? Something is wrong here. > > > Until I see a broad based study comparing QPCR with culturable and > total spore sampling that shows a better predictions of mold related > symptoms, I will continue use culturable and total spore methods. > > Based on the existing science, I see no scientific justification for > the extreme cost of QPCR analysis, except in very unusual > circumstances and then only along with culturable and total spore > sampling. > > Bob B > > > > > > > > > > > > > Wei Tang, Ph.D.Lab Director QLab5 DriveCherry

Hill, NJ 08003www.QLabUSA.com

[Guest guest]

When mold and spores are blasted they do not lose their toxicity. There are several recent articles on mold microparticles that conclude that such microparticles are more of a health problem that the spores. They do not lose their toxicity.

However, at least in Florida ... if the mold is dead and outside of the living space the claim that the job is done would be hard to argue.

Rosen, Ph.D.

QPCR Uses - A Question

Wei and others,Would QPCR analysis, or another technique, identify "mold" in the following situation? As I write this I'm increasingly aware of other analysis issues but I'll try to keep it fairly focused.Ice blasting was used to remove mold from OSB decking in a 2600 sq ft crawlspace. The remediator did not remove the significant residual dust that settled on the installed barrier over the soil. He/she claims removal isn't necessary because it's in the crawlspace and, besides, previous testing of such residue has always failed to identify the presence of mold. It has not previously caused him to fail PRV. The builder, who is paying for the work under his warranty, supports the remediator.The owner of the considerably valued home is highly allergic to mold, as is one of his children. He will agree with the remediator and the builder to leave the dust as long as it is dust and not mold residue. (It

should be removed anyway but that isn't the question here).The issue is the presence of triggers for allergic reactions, especially the proteins.The highly destructive nature of ice blasting may have rendered spore-based analysis unuseable. While ice blasting renders spores unidentifiable by either culturing or microscopy, does that necessarily mean the same is true for the allergy triggering proteins? Or the other components of the mold growth organism such as glucans, mycotoxins and enzymes. But I know of no studies either way.What methods of testing are available so I can reasonably state with any kind of certainty to my client that the residue from ice blasted mold remediation is "denatured?" Would the presence or absence of DNA (through PCR) be sufficient?Again, please ignore the several other issues involved and focus on whether or not the mold is "denatured" by ice blasting and how that

would be demonstrated.Carl GrimesHealthy Habitats LLC-----> Bob:> > Thanks for the info on PCR. It provided some insight and answers to > some of my questions and suspicions of PCR. Yes...Comparative > results are warranted. Moreover, the cost of PCR/QPCR tends to make > one collect, and rely on, fewer samples; and this is VERY risky when > assessing biologicals. Appreciate the input.> > -- > Geyer, PE, CIH, CSP> President> KERNTEC Industries, Inc.> Bakersfield, California> www.kerntecindustri es.com> > > > On 11/26/06 7:08 AM, "Bob s" <BobB@safety- epa.com> wrote:> > , > > "As many of you may know, several recent scientific studies have > looked at species analysis from culture testing and found

that they > are pretty much random when compared to DNA profiling of the samples > (called Quantitative PCR). Therefore culture testing for speciation > is of no value." > > Can you provide references for this statement? Since QPCR only > analyzes for some 30+ species, I have difficulty with this statement. > Further, culturable methods used in microbiological diagnosis of mold > infections in people have proven to be more than adequate from many > years. This QPCR claim is in direct conflict with medical research > methods. > > Further, at this point in time, there is no published scientific > evidence showing that QPCR has a stronger (higher correlation > coefficient) to mold related symptoms or disease, than any of the > other test methods. > > The published research history showing a relationship between > dampness, mold and respiratory symptoms

was well established in many > studies in the EU, almost 10 years ago. These studies were recently > duplicated in the US. However, this was not new information. > > There have also been studies in Germany and Canada that correlated > culturable levels with symptoms. Most other studies relate also > culturable levels to symptoms. This is why all of the mold exposure > standards in other countries are set in culturable levels. > > The only guideline on total spore concentrations is the AAAAI table. > However, note that this table drastically changed in 1992, when > AAAAI switch to Burhard from the Rot o Rod samplers. > > There is also no study that has proven that total spore sampling is > any better at predicting mold related symptoms than culturable > sampling. The key here is the strength of the correlation. P < > 0.01 or 0.05 are equivalent in scientific

power. Neither prove one > method is better than the other. > > Some people make the assumption that because hyphae or other mold > fragments can produce an allergic type reaction in a laboratory test, > that these products are a major source of the symptoms in people. > The fact is there is no data that show hyphae alone cause mold > related symptoms in people. It is extremely difficult to isolate > hyphae from the mold spores. It may be that you need 100 hyphae to > produce the same symptom as 1 mold spore. We simply do not know what > exactly is going on immunologically. > > This brings use back to QPCR. All analytic methods have a standard > analytical error. The SAE follows this rule. culturable<total > spore<<<< QPCR. > > The problem with QPCR is if you screw up, you are easily off by a > million. With the other analysis methods, you

may be off by a factor > of 10. > > I have worked in the drug industry with PCR and these analytical and > production errors are NOT uncommon. Lots of stuff gets thrown always > because of this. > > I have also seen QPCR results from clean, 1 year old wood, with no > visible mold growth, that the AIHA accredited lab reported levels of > 52,000,000. These same surfaces were negative on culturable > methods. > > The thing that really bothers me with the EPA pushing QPCR, is that > they do not do culturable and total spore parallels in ALL of there > research. > Why not? EPA goes through all the trouble to get an expensive QPCR > research project funded, and then they don't use historical methods > as a reference to the research? Something is wrong here. > > > Until I see a broad based study comparing QPCR with culturable and >

total spore sampling that shows a better predictions of mold related > symptoms, I will continue use culturable and total spore methods. > > Based on the existing science, I see no scientific justification for > the extreme cost of QPCR analysis, except in very unusual > circumstances and then only along with culturable and total spore > sampling. > > Bob B > > > > > > > > > > > > >

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[Guest guest]

Carl,

PCR looks at a snip of DNA from the specific mold species. As long as

the DNA snip is intact, it will reproduce it and indicate the presence

of a particular mold species.

However, in this case I would be weary of the results. Right now the

EPA is trying to develop some reference levels for mold and mold

products in normal homes.

Mold spores that are dry ice blasted and fractionated may not reflect

pieces of mold that can cause allergic reactions.

For example, how small of pieces do you have to break up hyphae into,

before they no longer produce an allergic reaction?

An ELISA test of the dust might be more meaningful.

However, given the allergic history of the family, I would recommend

removal of the dust and air washing of the crawl space. If the dust

came from the dry ice blasting, then it is not normally there and

should be removed as proper clean up. After all, you normally don't

leave construction dust piles at a building site. You remove the

construction debris.

It sounds like the building and remediator think that as long as the

visible mold is gone, they are done. However, proper cleaning of

residual debris is part of the job.

Bob

[Guest guest]

Steve,

Your comment is right on target. But note that molds produce both toxins as well as allergens. The blasting does not get rid of toxins either.

Rosen, Ph.D.

Re: QPCR Uses - A Question

In a message dated 11/27/2006 4:12:44 PM Eastern Standard Time, grimeshabitats (DOT) com writes:

What methods of testing are available so I can reasonably state with any kind of certainty to my client that the residue from ice blasted mold remediation is "denatured?" Would the presence or absence of DNA (through PCR) be sufficient?Again, please ignore the several other issues involved and focus on whether or not the mold is "denatured" by ice blasting and how that would be demonstrated.Carl,Denaturing of proteins involves changing the 3-dimensional (tertiary) structure of these molecules usually by destruction of cross-linkages. I don't think dry ice works to alter molecular structure at all. It justs physically abrades/pulverizes the larger pieces into smaller pieces of the same molecular structure. Some protein molecules

may be physically denatured but certainly not all.My question to you is why would you want to demonstrate to your client that "denaturing" of allergens has been effective at all? The burden of proof should be on those claiming that the CO2 treatment somehow has rendered the "mold dust" non-allergenic. But I'm sure you've already considered this.To answer your question, I think PCR testing could be used to show that molecules, in general, from the blasted mold were not atomized. The question about the tertiary structure and therefore the allergenicity of proteins would remain even if the DNA was intact for PCR detection. There may be some ELISA analytical methods that relate more directly to allergenicity of proteins. Again, why not just take the position that the allergens are there and let someone else try to prove otherwise?Steve Temes

__________________________________________________

[Guest guest]

Thank you, Bob. I appreciate the info and agree about the clean-up.

Unfortunately it's come to the point where the builder is saying

" Prove your claim. " I have no authoritative basis to support my

hypothesis and I know from experience that traditional spore analysis

by microscopy and culturing won't answer the question or address the

concern of the owner.

Carl Grimes

Healthy Habitats LLC

-----

> Carl,

>

> PCR looks at a snip of DNA from the specific mold species. As long as

> the DNA snip is intact, it will reproduce it and indicate the

> presence

> of a particular mold species.

>

> However, in this case I would be weary of the results. Right now the

> EPA is trying to develop some reference levels for mold and mold

> products in normal homes.

>

> Mold spores that are dry ice blasted and fractionated may not reflect

> pieces of mold that can cause allergic reactions.

>

> For example, how small of pieces do you have to break up hyphae into,

> before they no longer produce an allergic reaction?

>

> An ELISA test of the dust might be more meaningful.

>

> However, given the allergic history of the family, I would recommend

> removal of the dust and air washing of the crawl space. If the dust

> came from the dry ice blasting, then it is not normally there and

> should be removed as proper clean up. After all, you normally don't

> leave construction dust piles at a building site. You remove the

> construction debris.

>

> It sounds like the building and remediator think that as long as the

> visible mold is gone, they are done. However, proper cleaning of

> residual debris is part of the job.

>

> Bob

>

>

>

[Guest guest]

Why doesn't the bulider prove their claim that those "dust" is safe to be left there? Wei Tang QLab"Carl E. Grimes" wrote: Thank you, Bob. I appreciate the info and agree about the clean-up. Unfortunately it's come to the point where the builder is saying "Prove your claim." I have no authoritative basis to support my hypothesis and I know from experience that traditional spore analysis by microscopy and culturing won't answer the

question or address the concern of the owner.Carl GrimesHealthy Habitats LLC-----> Carl,> > PCR looks at a snip of DNA from the specific mold species. As long as > the DNA snip is intact, it will reproduce it and indicate the > presence > of a particular mold species.> > However, in this case I would be weary of the results. Right now the > EPA is trying to develop some reference levels for mold and mold > products in normal homes.> > Mold spores that are dry ice blasted and fractionated may not reflect > pieces of mold that can cause allergic reactions.> > For example, how small of pieces do you have to break up hyphae into, > before they no longer produce an allergic reaction?> > An ELISA test of the dust might be more meaningful.> > However, given the allergic history of the family, I would recommend >

removal of the dust and air washing of the crawl space. If the dust > came from the dry ice blasting, then it is not normally there and > should be removed as proper clean up. After all, you normally don't > leave construction dust piles at a building site. You remove the > construction debris.> > It sounds like the building and remediator think that as long as the > visible mold is gone, they are done. However, proper cleaning of > residual debris is part of the job.> > Bob> > > Wei Tang, Ph.D.Lab Director QLab5 DriveCherry Hill, NJ

08003www.QLabUSA.com

[Guest guest]

Carl,

Here are some alternatives.

I don't know how much water was involved in your case or how long it

was present. If there was a sufficient length of time you might have

grown some bacteria.

IF this is the case, you could have the dust analyzed for bacteria

endotoxins using the Limulus amebocyte lysate (LAL) test.

You could also gather some of the dust and have it animal skin tested

to see if it causes an allergic reaction.

Maybe the best way to deal with this is to have the homeowner hirer

someone to clean the residue off the plastic. They could then sue for

the extra cost.

Bob