Wakefield, O’Leary and Bustin

Below is an old post from 2007 when the Omnibus hearings were in full swing. It goes through the testimony of Professor Stephen Bustin and why it was so deadly to the MMR hypothesis. I thought now might be an ideal time to republish it.

On Day 8 of the Autism Omnibus proceedings, the MMR section of the hypothesis under examination (that thiomersal and MMR together cause autism) was examined. Just to briefly generalise about the MMR hypothesis –

It was hypothesised that measles virus (MV) from the MMR was travelling from the injection site, to the gut and then to the brain where it either a) causes autism or b) in conjunction with thiomersal causes autism. Wakefield claims to have found MV in the gut of several kids. Krigsman claimed to have replicated this work. They both used the lab of one Professor John O’Leary (Unigenetics IIRC) in Ireland.

So, on Day 8 of these proceedings Stephen Bustin came to the stand. Bustin is possibly _the_ world expert on the techniques used in the O’Leary lab that he claimed led to identifying MV in autistic kids gut and brain. The technique is called PCR. Not only does Bustin use PCR every day, he has 14 papers in the peer reviewed literature on PCR, over 8 book chapters and is personally the author of the _A to Z of Quantitative PCR._ which is considered ‘the bible’ of PCR. One of his papers has been cited over 1,000 times. Another has been cited over 500 times. He both organises and speaks at international PCR conferences (1934 – 37).

Basically, when it comes to PCR – this is the guy.

The first part of Bustin’s testimony concentrated on an explanation of PCR techniques and how these techniques were employed in a key study relied upon the week previous to make the MMR/Cedillo case: Uhlmann 2002. This paper describes the _exact process that Unigentics lab used to carry out their PCR work_ .

This is a mind bogglingly techie area so I’m going to try and stick to laypersons terms. Basically, the Uhlmann study is badly flawed. The key issues related to controls. Uhlmann (and a subsequent, as yet unpublished Walker poster presentation) didn’t use any.

a positive control is an essential control that tells you whether your assay is working, so what you would do is you would take the target that you’re interested in detecting and put it into a test tube and use your assay to detect it. If you don’t detect it, you know there’s a problem with your assay because it’s a positive control. If you do detect it, you know your assay is working. If you do this consistently each time, you know how efficient your assay is from day to day.

The positive control is simply something that tells you that your assay is okay.

Q And a negative control?
A A negative control is something very crucial. There you leave out your target, so if you don’t detect it then that means that there’s no amplification, which is what you want. If you do detect a positive in a negative control then you know there’s a problem with your assay because it should not be there, and you always get suspicious of any assay that gives you a positive result in a negative control.

So, back to Uhlmann:

Q And did Uhlmann provide the information necessary to establish whether these controls were working as expected?
A No. One of the surprising aspects of this paper is they give you very little information about how the assay was performed, about what the results actually were, and it really does not let you evaluate at all how reliable and consistent the results are.

Q Is there any discussion in Uhlmann about contamination?
A No.

Q Is this important?
A It is essential because obviously if you are trying to detect a very low copy number target and there is contamination around, and if you do not know whether there’s contamination around, then you can’t rely on your assay.

……

Q Now, did Uhlmann discuss how the RNA was handled?
A No. As I think I said one of the things about this paper is that it’s fairly unique in my experience, and it’s given no information at all about what actually was done. It actually tells you in outline what they did, where they got their samples from and that they prepared RNA, but it gives you no information whatsoever about, for example, the quality of the RNA the quantity of the RNA and how the different RNAs were extracted from different samples which they refer to.

Without being sure of how RNA is handled, it is impossible to rule out contaminants. i.e. that the samples are contaminated. And there’s more:

Q Did you also identify a mismatch between the measles virus sequences listed in the paper and the probes?
A Yes. This is, again, well, it suggests that there’s a problem with the probe design.

Q Now, regarding consistency and reproducibility did Uhlmann provide any data regarding amplification sensitivity or efficiency?
A No. I need to come back to what I’ve been saying several times now. There’s this lack of information that doesn’t allow you to evaluate this paper properly in terms of its validity.

And specifically regarding the Walker poster presentation:

….Now, as I tried to point out today I think virtually every expert in this case has referred to controls are essential. You always want controls of your samples. There’s no controls on this. So even though this is a poster presentation at the very least there should be a negative control on there to show that the PCR in the negative control hasn’t worked.

We don’t have that information. So this immediately invalidates these results because we can’t now say whether these are genuine or not because there’s no negative control there. So that’s a real problem….

Q Unless these issues are resolved would you have confidence at least in what’s been presented from the Walker lab?
A I can’t have any confidence because there’s actually no results I can evaluate without referring to a negative or a positive control, and these don’t give them to me.

So basically, Mr PCR – the guy who literally wrote the book on PCR – thinks these two things – the Uhlmann paper and the Walker poster presentation – are essentially useless.

Lets also not forget that these two items describe the exact methodology that Unigentics – O’Leary’s lab – used to state that Wakefields/Krigsmans and a multitude of private cases had MV in their samples.

Oh yeah – and as for the old crapola about the two clinical studies done thus far not repudiating Wakefield et al because the look at blood, not gut, Bustin states emphatically:

this is not an assay that is at its limits so this should be easily detectible, and it also means that if you’ve got that much measles virus in a gut sample it probably is in other cells as well and you should be able to detect it, for example, in blood.

In blood.

Turning to Unigenetics itself, as part of the failed UK litigation against MMR, Bustin was asked to look at the lab and samples that the Unigentics team had worked on. For this work he put in an astonishing 1,500 hours of work.

Now, Professor O’Leary’s own controls tell us that this should have been shifted upwards because this is much poorer quality RNA. The evidence from his own data is completely clear. There’s no such shift. This must mean that whatever this is is a contaminant that has been introduced after the sample has been formalin-fixed.

So by definition this cannot be part of the original biopsy because if it had been it will have shifted upwards.

Ouch. But the next one is a body blow to the entire MMR hypothesis.

…if you have a reference gene in that sample that is a cellular reference gene you should detect it if the RNA is of good quality.

If you don’t detect it there’s something wrong with the RNA. As Professor O’Leary’s SOP states, if we can’t detect the GAPDH we shouldn’t use the sample for analysis, which makes perfect sense.

Now, it happens that Professor O’Leary did use those samples for his analysis, and that’s why I was able to then hopefully identify what the contaminant is.

To summarise: O’Leary’s RNA was poor quality. There is no reference gene. There is something wrong with the RNA. O’Learys lab SOP (Standard Operating Procedure) states that in the events of this happening, they shouldn’t use the sample for analysis. But they did use it. They used contaminated RNA for their analysis. And Bustin has identified exactly what the contaminant was.

But first, what is _definitely not_ ?

If you detect a target that is apparently measles virus in the absence of an RT [like this one] step by definition it can’t be measles virus because it has to be DNA [measles virus does not exist as a DNA molecule]. It’s a very simple concept. At least it is to me. It’s not to everyone else.

Whatever it is that O’Learys lab is picking up in their lab tests, it cannot possibly be Measles virus. No RT step was taken:

What I immediately observed was that they had forgotten to do the RT step

No RT step means it’s DNA. Measles virus does not exist as a DNA molecule. That’s simple medical fact. Bustin later summed up:

So all of this evidence suggests very, very strongly that what they are detecting is DNA and not RNA. Because measles virus doesn’t exist as a DNA molecule in nature, they cannot be detecting measles virus RNA. They are detecting a contaminant. All of the additional evidence, from the nonreproducibility by Professor Cotter of the same samples that Unigenetics analyzed to the analysis of the data where there are discordant positives, where the negatives came up positive, suggests very, very strongly to me that there is a lot of contamination in the laboratory, which is not unusual, but they have not handled it very well in how they have troubleshot their problems.

So I have very little doubt that what they are detecting is a DNA contaminant and not measles virus, and I do not believe there is any measles virus in any of the cases they have looked at.

And just as an added kick in the teeth:

Q Now, we know that cerebral spinal fluid samples were sent from Dr. Bradstreet to Unigenetics for testing. Do the same concerns you’ve outlined here apply to that testing?
A Yes. Exactly the same concerns would apply to that.

This testimony exposes the MMR hypothesis as totally dead in the water. What a waste of time, money and health.

Elsewhere

Arthur Allen in the Huffington Post
Autism Diva