Fellow Sciblogger Grant Jacobs and I have both recently posted on the BMJ’s editorial ‘Wakefield’s article linking MMR vaccine and autism was fraudulent’. The editors left us with plenty of food for thought, including:
‘What of Wakefield’s other publications? In light of this new information their veracity must be questioned. Past experience tells us that research misconduct is rarely isolated behaviour’
What of his other work? Indeed, the Lancet paper was just the first in a series a papers attempting to link autism with measles. Last year I gave a talk at the NZ Skeptics annual conference about his other papers which used ‘proper’ science to shore up this link. It was entitled ‘The (bad) science behind the MMR hoax’. I think you can probably see where I’m going with this….
One of the bits of ‘proper’ science Wakefield used was the polymerase chain reaction (PCR). For those who don’t know how PCR works there are some video’s online. PCR is a fantastic technique used to amplify very small amounts of genetic material to generate over a billion copies. In a nutshell this means PCR can take something that is undetectable and make it detectable. As you can probably imagine, one of the downsides of such a sensitive technique is that it is very easy to contaminate, so proper controls are really important. But I’ll get back to that in a minute.
One of the crucial things needed to carry out PCR is a set of very specific ‘primers’ which recognise the region of genetic material that you want to amplify. You need primers to each end of the region of interest and then, as if by magic*, PCR amplifies the bit between the primers. So the important things to remember are:
1. The primers need to be specific so that they only amplify what you are targeting and nothing else.
2. You have to be very very very careful not to contaminate the reaction.
Which brings me to controls. When carrying out a PCR reaction, alongside the samples you are testing, it is crucial to include the following:
1. A negative control which has water in place of any target genetic material. This will tell you whether you have had a contamination problem or not.
2. A negative control which has control genetic material that does not contain any of the target sequence. This will tell you if your primers are specific enough.
3. A positive control which has genetic material that does contain the target sequence. This will tell you if your reaction has worked.
So, you have your samples and your controls, the PCR machine has done its dash and you are left with a little tube filled with billions of copies or the target sequence (or none if the sample was negative….). This can then be visualised by gel electrophoresis and you are left with something like the picture below.
Lane 1 contains a size standard, lane 2 is the negative control containing no genetic material, lane 3 is the negative control containing no target sequence**, lane 4 is the positive control containing the target sequence and lanes 5 and 6 are our unknown samples (which in this case are all positive). It is important to say here that very rarely would you see an actual gel published in a paper***. Most results are just described as the number of positive or negative samples. This is important as it leaves the reader assuming the correct controls were done. But it doesn’t end with bands on an agarose gel. The amplified genetic material can then be sequenced to confirm it is the correct thing. And if the claims you are making are wide-reaching and/or controversial then sequencing is exactly what should be done.
So back to Wakefield’s bandwagon. What Wakefield hypothesised was that exposure to the measles virus in the MMR vaccine was a factor in the emergence of his so-called ‘autistic colitis’ and that genetic material from the measles virus would be found in patients with the disease but not healthy controls. He supervised a PhD student Nick Chadwick to investigate. The first paper they published (in January 1998) was in the Journal of Virological Methods reporting a ‘rapid, sensitive and robust procedure’ for amplifying measles RNA. In August 1998 they published a second paper describing the use of the procedure to look for measles virus in samples from patients with inflammatory bowel disease (IDB). They state:
‘These results show that either measles virus RNA was not present in the samples, or was present below the sensitivity limits known to have been achieved.’
They then went on to look at the children reported in the, now retracted, Lancet paper (that is, the ones with ‘autistic colitis’). Wakefield never published these results but Nick did write up his PhD thesis in 1998****. Brian Deer has put the relevant information from the thesis here. I’ll spare you the suspense. Nick concludes:
‘None of the samples tested positive for measles, mumps or rubella RNA, although viral RNA was successfully amplified in positive control samples‘
Despite this negative result from 1998, Wakefield then appears as senior author alongside a team of Japanese researchers in a paper published in April 2000 in the journal Digestive Diseases and Sciences where they report the detection of measles virus:
‘One of eight patients with Crohn disease, one of three patients with ulcerative colitis, and three of nine children with autism, were positive. Controls were all negative. The sequences obtained from the patients with Crohn’s disease shared the characteristics with wild-strain virus. The sequences obtained from the patients with ulcerative colitis and children with autism were consistent with being vaccine strains.’
That’s three of the children Nick had already found to be negative.
In 2002 Wakefield then published another bigger study of children suffering ‘autistic colitis’ with a team from Ireland. They reported:
‘Seventy five of 91 patients with a histologically confirmed diagnosis of ileal lymphonodular
hyperplasia and enterocolitis were positive for measles virus in their intestinal tissue compared with five
of 70 control patients.’
So how did they get any positives when Nick’s results were all negative? Yasmin D’Souza and colleagues at McGill University in Canada published a very nice paper in 2007 in which they compared the primers used by both the Japanese and Irish groups with their own primers for the measles virus on a range of IBD and control intestinal biopsy samples. Any positive samples were verified by sequencing. And the results? The primers used by Wakefield and colleagues weren’t specific for measles virus. In fact, the amplified fragments were found to be of mammalian origin. What this means is that human samples should all be positive. This begs the question: why were only 5 of the Irish team’s controls positive?! Were they not human?
So what happened when D’Souza tried genuine measles specific primers? Surprise, surprise, they
‘failed to demonstrate the presence of MV nucleic acids in intestinal biopsy samples from either patients with IBD or controls’
In another paper D’Souza and colleagues also tried their measles specific primers on samples from over 50 autistic children. Their findings?
‘No sample from either autism spectrum disorder or control groups was found to contain nucleic acids from any measles virus gene.’
So, does that count as research misconduct or incompetence? I would argue Nick’s PhD thesis suggests the former.
*By which, of course, I mean beautifully elegant science…..
**Don’t be alarmed by the faint band, this is just the background genetic material.
***Of course the reviewers could ask to see electronic copies of the original gels but this is very rare. Saying that, I know of at least one case where a post-doc was found to have completely fabricated her entire two year project when a reviewer asked to see the original data and it was found that she didn’t have any.
**** and is now Dr Chadwick…