Thursday, December 29, 2016

What has Andy Wakefield done for science?

With the resurgence of Andy Wakefield through his "documentary" Vaxxed, his prior work has come back into focus. It's clear that his work as a researcher contains many errors of omission and commission, but why is the scientific community certain that something nefarious happened in his prior work? There is of course the excellent work from Brian Deer showing how the paper published in The Lancet was fraught with fraud and undisclosed conflicts of interest (being paid nearly a half a million pounds to show that the MMR is dangerous is kind of a conflict of interest). That investigation led to Andy losing his medical licence in the UK. However, much of the focus has been on the 1998 Lancet paper and not as much on his other work. I'll discuss one of his other papers here as it is incredibly clear that at best, inept data analysis and sample handling was done.   

In 2002, Andy was an author on a paper entitled "Potential viral pathogenic mechanism for new variant inflammatory bowel disease" published in Molecular Pathology (the PubMed Central deposit can be found here). The paper uses qPCR detection of the Measles virus using the Taqman chemistry (see image below). 

This is how Taqman qPCR works.

The results of the paper look normal to the lay person; however, if a researcher who is familiar with qPCR looks at the raw data from the paper, an enormous problem is evident. I rarely share youtube videos; however, this video from C0nc0rdance explains qPCR and why the issue is such a huge deal.

For those who don't want to watch the video, the problem is simple. qPCR is done using a thermocycler that uses a laser to detect a fluorescent target and is linked to a computer. The software on the computer automatically does calculations for determining what is positive and what is not. One of the calculations that is done is the threshold for determining what is considered a positive reaction and what is not. In the 2002 study, the threshold was manually lowered so that samples that should have been negative came up as positive. These samples were all after cycle 30 which means that it was a small quantity of material being detected. Additional analysis of the raw data by Dr. Bustin (an expert on qPCR) found that these were false positives due to contamination. So we have two major issues to contend with here. First the samples were contaminated (qPCR is sensitive to contamination so extra care has to be taken to prevent this). Second, the data was improperly analyzed and the Ct threshold improperly lowered so that these samples became positive. At best, this is poorly conducted science that should be retracted. At worst, if the threshold was knowingly altered to generate positives, this would be scientific misconduct. It's not likely that the contamination was intentional as it was in low quantities (why spike a sample with an amount that would look like contamination rather than spike with an amount that would clearly be positive?) and some of the researchers involved in this study published a subsequent study that found no measles virus when the experiment was replicated (three independent labs did the tests on each of the samples), so the adjusted analysis was probably done in ignorance rather than malice.

But this paper (and several others) did have a positive impact on the scientific community. It lead to the creation of the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE). These standards dictate what the minimum amount of information needs to be included with a paper using qPCR data. This includes information on how the samples were extracted, how the test was validated, what controls were used with the test, and how the data were analyzed. Many of the top journals have adopted these guidelines and many researchers follow them even if it isn't required for publication (it's just good science). 
Information needed to fulfill the MIQE guidelines.
So even though Andy Wakefield is at best an incompetent researcher (and a con artist at worst), he has done something good for science. As a result of his work (and the work of others), the standards for qPCR reporting have been increased so that the results are standardized across disciplines and publications. It does bring up an interesting question, how many of Wakefield's previous studies are faulty and what should be done about them. In this case, a correction should have been submitted, at the very least, when it became clear that the data analysis was not done properly and that the resulting conclusions are suspect. 

However, one thing is clear. Andy Wakefield is not the virtuous crusader for the truth that has been unfairly attacked by the forces of evil. Despite the narrative that he tells, his work speaks for itself. It is full of errors and improprieties that make it all suspect. His actions are what led to him being shunned by the scientific community. Andy shouldn't be blaming anyone but Andy. 

Saturday, December 3, 2016

Genetic modification of Influenza A virus reveal a novel vaccine production strategy

Researchers have developed a new type of vaccine. By genetically modifying the genome of Influenza A virus to require a non-standard amino acid (Ne-2-azidoethyloxycarbonyl-L-lysine which has been used in synthetic biology previously) that some microbes use. What makes this amino acid unique is its codon (three base RNA sequence that signals what the amino acid should be). In most organisms, the codon used (UAG) is actually what is known as a stop codon (it stops production of the protein chain), so this means that any gene that contains that codon will stop prematurely unless grown in an organism with that non-standard amino acid. A lot of work went into finding places in the viral protein that the non-standard amino acid could be inserted without interfering with the function of the protein. Once several sites were identified, the researchers grew the virus in kidney cells that had been genetically modified to use the non-standard amino acid. The end result was genetically stable progeny viruses that required the non-standard amino acid.

When the altered influenza virus was injected into mice, it did not replicate and the mice developed a strong immune response to the virus. Researchers were able to safely inject the mice with a dose of the modified virus that was about 100,000 times the dose of the wild-type virus that would kill half of the mice injected (LD50). The modified virus also interfered with the wild-type influenza virus in co-infections of the two.  

The implications for this development could be astounding. With this, researchers could develop a vaccine that uses a fully infectious virus that has been modified to need this amino acid to replicate. However, caution is warranted at this point. A lot of work still remains to develop this into a functional vaccine. It must go through further trials to see if it works in primates and then there are all the human clinical trials that are needed. This work is promising and certainly should be pursued further.

This negative stained transmission electron microscopic (TEM) image shows recreated 1918 influenza virions that were collected from supernatants of 1918-infected Madin-Darby Canine Kidney (MDCK) cells cultures 18 hours after infection. Photo credit: Cynthia Goldsmith courtesy of the CDC.

Friday, December 2, 2016

Poinsettias and pathogens

Poinsettias are a very interesting plant. Originally, this plant grows as either a small shrub or a tree in its native range (Mexico). However, the little potted poinsettias that can be bought at the store never grow close to that size. There is a good reason for this: the potted poinsettias are infected with a pathogen known as a phytoplasma. Phytoplasmas are small bacteria that lack a cell wall and are limited to the phloem tissue within plants. They are spread by vegetative propagation, grafting and by sap-sucking insects that feed in the phloem. Normally phytoplasmas are associated with severe disease in plants by altering the structure of plants, such as causing the petals of flowers to develop into leaves instead of petals. One such disease is aster yellows which infects over 300 different plant species in 38 families (see picture below).
Aster yellows on the Purple coneflower (Echinacea purpura). Via wikipedia
But what does this have to do with poinsettias? It turns out that the phytoplasma that infects the potted poinsettias also causes stunting, but doesn't cause severe distortion of the leaves. Not only is the plant stunted, but it produces additional branches where normally the plant has a single branch. The trait has been used since the 1920's to sell poinsettias during November/December in North America. This trait was found to be transferable by grafting but could be lost when the plant was subjected to heat treatment or tissue culture techniques. It was thought that another pathogen that is common in poinsettias was the cause of the stunting, Poinsettia mosaic virus, but it wasn't until the 1990's that a phytoplasma was shown to be the causal agent of the stunting. Previous work found that the virus wasn't completely associated with this trait as plants without the virus developed stunting and free branching. This article contains further information on the history of poinsettias and the work to determine why commercial poinsettias are stunted and free branching. This article discusses how to care for poinsettias.

A poinsettia tree in Mexico. Via Petal Passion

A potted poinsettia. Via pixabay.

The phytoplasma infecting poinsettias is a perfect example of how not all pathogenic organisms are bad and that they might be of benefit. This is certainly the case in the ornamental industry as it is the basis of the entire ornamental poinsettia industry.