Negligent? Sloppy? Intentionally so? You decide.

Andy Wakefield published his "research" that was used by the anti-vac
liars to frighten people into not vaccinating their children for MMR. As
a result, there were numerous cases of these vaccine preventable
diseases, deaths and most likely permanent disabilities.

Now, new research shows why Wakefield's research found what it did. He
did not prepare his samples properly, and this can result in false
positives.

From: http://www.badscience.net/?p=313

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Ben Goldacre
Saturday October 14, 2006
The Guardian

Think back into the mists of MMR: in 2002, Professor John O’Leary’s
group in Dublin reported finding measles virus in the intestine of
children with autism and bowel problems. The anti-MMR movement were
almost delerious with excitement, and so were the media. Andrew
Wakefield, working with Kawashima et al in Japan, had already reported
finding measles virus in blood cells in similar children.

What if they were mistaken? How would you know? Well.A major paper
published in the leading academic journal Pediatrics this month
strongly suggests that these earlier results were in error, false
positives. This study has been unanimously ignored by the media: it has
been covered, by my reckoning, in one Reuters piece, and in one post on
the lead researcher’s boyfriend’s blog. Nowhere else. Although the
papers can find space for multiple stories about unpublished “studies”
on how you should buy fish oil pills for your children. I digress.

This new MMR study, by D’Souza et al, replicates the earlier
experiments pretty closely, and in some respects more carefully, in 54
children with autism (80% also had gastrointestinal symptoms), and 34
controls. All but 6 had received the MMR vaccine.

All these studies, old and new, used PCR, the same process used in
“genetic fingerprinting”. PCR works by using enzymes to replicate
RNA, so you start with a small amount in your sample, but then it is
“amplified up”, copied over and over again, until you have enough that
you can measure and work with it.

Beginning with a single molecule of genetic material, PCR can generate
100 billion similar molecules in an afternoon. Because of this, the PCR
process is exquisitely sensitive to contamination - as numerous innocent
people languishing in jail could tell you - so you have to be very
careful and clean up as you go. One substance used to prevent this
contamination is called “UNG”. As an example of their
meticulousness, the new study used used 50 times more UNG than the
original O’Leary paper, because research has since shown that you need
these higher concentrations, to prevent contamination.

The researchers were also careful to use the very same primer
sequences for the measles virus genes as their predecessors. Primers
are what tells the PCR process where to start and stop copying RNA,
and they are what picks out the genetic material you are hunting for.

The results were striking. Firstly, using the Kawashima primer pairs,
they simply got negative results, where Kawashima et al reported
positive results. Go figure.

The replication of the O’Leary work was more interesting. Looking only
at the results of the PCR, at first it looked like the O’Leary primers
did indeed produce RNA strands that matched measles virus: the primers
designed to pick out measles virus found and amplified up some genetic
material. However, when they looked more carefully at the size of the
strands, and the “melting curves”, and then actually sequenced the
genetic material, they discovered that what had been amplified was not
actually from measles virus at all: they were false positives. The
original O’Leary paper did not pursue these extra “double checking” steps.

The authors are are quite clear: there is good reason to suspect that
the earlier studies produced false positive results, because of
suboptimal contamination control, and because the O’Leary primers can
accidentally amplify bits of normal human RNA. Let’s be clear: this is
absolutely not about criticising individual researchers. Techniques move
on, results are sometimes not replicated, not all double-checking is
practical: what is odd is that the media rabidly picked up on the
original frightening data, but has completely ignored the new reassuring
data. Too many fish oil pill stories, perhaps.

So how could an anti-MMR campaigner criticise this new study? In two
ways. Firstly they might take up minor differences in PCR technique:
although the onus would be on them to explain why these minor
differences invalidate the results. Secondly, they might say: for the
Kawashima blood cell replication, okay, fair enough; but O’Leary found
measles virus in gut biopsies, not blood cells… this study, they might
say, should have looked at gut biopsies.

This is interesting. Firstly, people currently argue that it is
unethical to take gut biopsy samples simply to refute a scare (I
disagree, now that people are dying from diseases preventable with MMR,
but I guess we all have our opinion). And there is no reason to imagine
that measles RNA should persist in gut only, and not blood. But lastly,
and most crucially, remember: this new study found just the same
positives identified by the O’Leary primers, but checked, and found that
they were apparently false positives. This is likely to be just as true
in gut as it is in blood.

This ignored negative finding is not even an isolated event. Afzal et
al performed a similar replication study, and also reported negative
results, and were also ignored. So in a week where newspapers found
room for even more “research” stories promoting fish oil pill
manufacturers, this study - of major significance, published in a major
academic journal, on a topic of major interest, on a topic where “more
research” was “demanded” - was ignored. In a country where unpublished
and unfinished “new research” on the dangers of MMR can become headline
news in almost all the broadsheets; in a country where MMR is still only
accepted by 83% of parents; this is contemptible.

References:

The paper (sorry, full manuscript needs cash or academic sub):

pediatrics.aappublications.org/cgi/content/abstract/118/4/1664

The Reuters piece which no-one picked up:

http://www.emedicine.com/news.asp?na...cine%2 0Today

A fun looking video on PCR and MMR (not watched it all myself yet):

bartholomewcubbins.blogspot.com/2006/10/bc-on-autism-17-pcr-primer.html

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He was either sloppy accidentally, or intentionally. His results fit his
clients needs.