Controls Versus Contamination

[alex3619]

One Phoenix Rising member has suggested I update my comments on XMRV since the following post:

http://niceguidelines.blogspot.com/2010/12/my-take-on-cfs-xmrv-as-of-today.html

I still agree with these points. I would like to add the following comments:

1. This commentary includes in the heading "as of today" for a reason. The science can change very rapidly. It only takes one really good new scientific paper to do this.

2. People have commented that the epidemic ME outbreaks do not match our understanding of XMRV lifecycle. On the contrary they are a good match using the two hit hypothesis which has been repeatedly discussed on Phoenic Rising. Indeed I have discussed the two hit hypothesis on another forum since before we even knew about XMRV.

3. All of the contamination papers do one thing: they raise a possibility of contamination. None of them prove contamination in all the studies that could find XMRV. Indeed, the fact that some studies showed mouse DNA contamination demonstrates that careful screening can detect such problems. Contamination with live XMRV is another issue, which I have also discussed before. See additional commentary below.

4. None of the contamination papers explain why the control groups in any of the studies are not massively contaminated. Again, see additional commentary below.


Contamination Findings

All the contamination studies do is show that contamination could be a problem. This is already known to researchers. The mouse contamination issue has been so well dealt with in various places that I am not going to comment further. The idea that samples could be contaminated by ... XMRV, deserves further comment.

The two CROI papers by Coffin et. al. that discuss ancestral viruses and recombination events show that XMRV might have been from recombination of viruses from nude mice used to develop the 22Rv1 cell line:

http://www.retroconference.org/2011/Abstracts/42569.htm
http://www.retroconference.org/2011/Abstracts/42508.htm

XMRV was previously shown to match the sequence of a combination of two endogenous MLVs, but showing that these viral precursors exist in some lab mice that are used for xenografts is new.

The key point from the CROI conference is additional confirmation that XMRV is a real infective virus, not that it might be a lab artifact. The claim that XMRV might have arisen in these cell lines due to recombination is possible. It is also possible that XMRV may be present due to contamination from people (or other sources) who carry the virus.

The argument that XMRV could have arisen only once in the 22Rv1 cell line is probabilistic. It might arise once, but it is highly improbable it could have arisen several times. For the probability to be a viable tool, this has to be purely random. I very much doubt that is a purely random event, given that it is to MLV's evolutionary advantage that such crossovers occur.

This paper has also not shown that XMRV did not arise from recombination in the early 1900s (or any other time) either - the arguments against them are essentially the same as the arguments against the Lombardi et. al. findings, except Lombardi et. al. have way more evidence and XMRV has been found in many labs in controls and patients.


Controls Groups

The purpose of control groups is either to perform a check on the experiment to highlight problems, or creating a benchmark to compare the results to. If XMRV were a contaminant you would expect that it would be present in the same percentage in patients and controls. While it might be argued that the patients are more likely to be contaminated due to extra handling, could this be true for lab after lab, sample after sample?

The overwhelming probability is that the low percentage in the control groups, plus much higher percentage in patients, completely rules out all contamination hypotheses. Unless the contamination hypotheses can explain this, they are just hypotheses.

What this study does have is a possible origin for XMRV, a possible mechanism for its creation, and the presence of XMRV. It is enough to make us stop and think, it is not proof of contamination.

What about the zero zero studies - studies in which zero or close to zero XMRV is found in both patients or controls? These tell us several things too.

First of all, if contamination is such a problem, how come these studies are not routinely contaminated? The obvious argument is of course that only those labs that have an XMRV contamination problem will produce positive XMRV findings - but this is both not proven and many of the labs never used the 22Rv1 cell line.

If this is a real virus, which the evidence strongly supports, and it is present in the population, which is likely, then why cannot these studies discover a background rate? There seem to be only three likely reasons. First, the background rate might be very low and with limited geographical distribution, especially if the cell line origin hypothesis is correct. Second, while this cell line might be pumping out virus particles they might have been contained. Third, these zero zero studies might not be finding XMRV because assumptions made during design and testing may not be valid in a human population infected with XMRV. Which answer is correct is still debatable.

Other MLVs including polytropic MLVs may also be an issue. None of the contamination papers have really examined these. While antibodies can cross react with other antigens, the presence of viral proteins indicates a replicating virus.

In my view the balance of evidence is still that XMRV is a real virus, and that it is present in the general population at a low rate (maybe up to 10%), a minority of prostate cancer patients and many ME/CFS patients. Whether it is primarily causal for CFS is still to be determined, there is a distinct possibility that ME/CFS is due to an immune response to fight the retroviruses.

Thank you for reading this far, even if you disagree with me, Alex Young

Recommended Reading:

http://www.mecfs-vic.org.au/sites/www.mecfs-vic.org.au/files/Article-2010Mikovits-Detection.pdf

While you are reading you might like to read my post on writing scientific articles, which is hidden away in the projects section (okay, okay, a shameless plug):

http://forums.aboutmecfs.org/showth...ely-we-can-have-medical-and-scientific-impact.

 

[SOC]

Thank you so much for this, Alex. While I have a research background, my field is not biomedical sciences so your summary of the current XMRV origin and contamination reports was extremely helpful to me. I imagine it is equally valuable to many others here at PR.

Many, many thanks for your hard work and masterly summary.

 

[acer2000]

Thanks alex,

One thing I think these "contamination" studies also fail to address is that neither 22Rv1 or DU45 cell lines were used by the original Lombardi study. They used LnCAP. Its certainly possible that LnCAP could be susceptible to contamination - but until this is directly tested for in an experiment and published, these theories about contamination causing the results in the original study are rather circumstantial.

The other confounding factor here is that they are using human prostate cancer cell lines to do the culturing in. The very disease they think is associated with the virus. That creates kind of an awkward situation. Even you find the virus in the culture cells before you add CFS patient blood - you can't really be sure its purely lab artifact because it could have been in those cells because it caused the cancer in the patient the cells came from in the first place. I suppose with 22rv1 they went back and found older cells before they had passed them through mice and found those cells to be negative, so that seems to help the case for that cell line. But I don't know that the same can be said for the other cell lines.

Of course all of these things could be true as well. That these human cancer cell derived lines may have XMRV them, that XMRV got into the human population and caused cancer, and that it also causes other illnesses like CFS. Just because they find it in a cell line, doesn't mean it doesn't exist in and can't cause illness in actual humans. In fact, it perhaps gives more credence to that idea, since it clearly can grow in human cells.

Does anyone know the origin of LnCAP? Did it also pass through mice? Was it explicitly tested -ve for XMRV before it was combined with patient blood in culture? I think I read that it was, but I couldn't locate the reference..

 

[alex3619]

Hi acer2000, LnCAP dates back to 1982

http://cancerres.aacrjournals.org/content/43/4/1809

Bye,
Alex

 

[Marg]

If this is a real virus, which the evidence strongly supports, and it is present in the population, which is likely, then why cannot these studies discover a background rate? There seem to be only three likely reasons. First, the background rate might be very low and with limited geographical distribution, especially if the cell line origin hypothesis is correct. Second, while this cell line might be pumping out virus particles they might have been contained. Third, these zero zero studies might not be finding XMRV because assumptions made during design and testing may not be valid in a human population infected with XMRV. Which answer is correct is still debatable.

Other MLVs including polytropic MLVs may also be an issue. None of the contamination papers have really examined these. While antibodies can cross react with other antigens, the presence of viral proteins indicates a replicating virus.

In my view the balance of evidence is still that XMRV is a real virus, and that it is present in the general population at a low rate (maybe up to 10%), a minority of prostate cancer patients and many ME/CFS patients. Whether it is primarily causal for CFS is still to be determined, there is a distinct possibility that ME/CFS is due to an immune response to fight the retroviruses.

Thank you Alex for making things so much clearer for me. I never did understand the contamination issues. I feel a lot better about the whole ball of wax after reading your post, really great job!

 

[acer2000]

One of the things I also wonder is whether they can run an assay for MLV reverse transcriptase? Or even a general reverse transcriptase? I know that enzyme is specific to retroviral infection (ie the body doesn't make it otherwise), but perhaps it would be an indirect measure of whether they are headed in the right direction. If such activity was found in patient samples that hadn't been cultured with potentially contaminated reagents or cell lines, that would be telling.

Also, the thing I always am saying is why are they not doing tissue biopsy and checking that? The monkey study showed that XMRV isn't really in the blood reliably. It did, however, identify about 5 other tissues that showed viral persistence. Someone should do a study on CFS patients and look in those places.

 

[alex3619]

One of the things I also wonder is whether they can run an assay for MLV reverse transcriptase?
Also, the thing I always am saying is why are they not doing tissue biopsy and checking that?

Hi acer2000, identification of reverse transcriptase would indeed show the presence of a retrovirus. I think we might be hearing more about this soon - its so obvious.

I too have been calling for biopsies. This debate would be so much simpler if we had biopsy results as well as blood results.

Bye
Alex

 

[kurt]

3. All of the contamination papers do one thing: they raise a possibility of contamination. None of them prove contamination in all the studies that could find XMRV. Indeed, the fact that some studies showed mouse DNA contamination demonstrates that careful screening can detect such problems. Contamination with live XMRV is another issue, which I have also discussed before. See additional commentary below.
4. None of the contamination papers explain why the control groups in any of the studies are not massively contaminated. Again, see additional commentary below.

...

The overwhelming probability is that the low percentage in the control groups, plus much higher percentage in patients, completely rules out all contamination hypotheses. Unless the contamination hypotheses can explain this, they are just hypotheses.


Thank you for reading this far, even if you disagree with me, Alex Young

Alex, I agree completely, the contamination papers only raise the possibility of contamination. By the same measure, the WPI studies only raise the possibility of infection with XMRV. Neither side has conclusively proven its point. Early studies simply provide 'proof of concept' of the points made, until many further studies characterize the situation in a broader fashion we don't have all the needed information to draw conclusions.

As for your comment that difference between patient and control samples completely rules out contamination...I do disagree with that. In these early studies, including the WPI study, convenient samples were used. This means the source of control samples was different from the source of patient samples. They were handled and treated differently, etc. In fact Mikovits said at one point they had to test the patient samples many more times than the controls to get the positives to show. But then she said they had controls in each batch. I don't know enough to make sense of what she said but know outside researchers see the possibility of contamination.

Incidentally, differential collection and/or testing is also true of many if not all of the 0/0 studies so far as I am aware. They take convenient samples of both patient and controls, and they come from different labs and sources. But with 0/0 findings I guess that does not matter much in terms of contamination, but there could be systematic problems in samples used on both sides at this early stage. Maybe in the future there will be a study where controls and patient samples are collected and storied identically, and tested an identical number of times, then if blinded tests can pick out the CFS patients, the contamination theory will have a different problem. But even then a testing artifact is still possible, I heard or read somewhere when I was studying PCR testing a year ago that there can be interactions between reagents and specific genes in a test population.

This is why I promote the idea here that we have to let the science run its course before drawing conclusions. And also, this is why I have been more skeptical than others. Although my research area was not biology, I did work a lot with human subjects professionally as a researcher before getting CFS, and the promotion of XMRV by WPI raised a warning flag in my mind right away. That is just not good science, getting hopes up so early in a new discovery is bad form in any scientific field.

 

[kurt]

I apologise in advance for the possible gross stupidity of this question, but it is bothering me. There is now a suggestion that XMRV is a man-made virus or "contaminant". Yet this "contaminant" has been shown to be infectious in human cell lines - or did I pick that up wrongly ? Even if XMRV is a man-made virus, is there anything to stop this thing actually causing a subset of "CFS" cases ? I assume most reasonable people accept "CFS" results in compromised immune systems - is there an obvious scientific reason why this man-made virus could not account for some CFS cases against such a background ?

If XMRV had its genesis in the lab, then the greater question is whether or not it has 'escaped' into the human population. There was evidence presented at CROI that is unlikely, as there is more strain diversity in the human cell lines than in the humans who actually tested positive. Based on evolutionary theory, this suggests that the positive tests are likely to result from contamination, as a true infection in the population would by now have far more genetic diversity than the lab cell lines.

But I think you are also making a larger point with your question, could XMRV theoretically cause CFS in a subset if it got out of the lab? I think that is highly unlikely based on all the evidence, but of course others might disagree. Why I think it unlikely, other studies have shown that there is a natural human cellular defense mechanism that easily defeats XMRV in a live human. The persistence of XMRV in cell lines does suggest it might be possible though, perhaps if a lab tech got the virus injected into her blood in some accident. But what would then happen? We don't know, the monkeys who were given huge doses of XMRV did not get sick, but they also did not have long incubation periods, nor very long lives (sadly, for them).

We do actually have a small subset of CFS patients who harbor a retrovirus, HTLV. This was proven (and then ignored) by WPI in their Virachip study in 2009. But of course in that case the retrovirus, which has been associated with at least one non-CFS disease, is not likely causing the CFS in the general CFS population, otherwise more of the patients would have been positive.

 

[kurt]

Thanks Kurt. Yes, I've seen the diversity issue brought up a number of times.

I think you're referring to APOBEC3 as the protein which effectively blocks XMRV replication. I wonder if there are genetic polymorphisms of this protein, or illness mechanisms in other diseases which might suggest a compromised APOBEC3 function ? That would give it a potential advantage if present in some CFS patients.

Yes, the hypermutation situation, that is the protein. I don't know if that mechanism has SNPs, we really need some full genomic profile studies in CFS. That in my opinion is where our research dollars should head right now, full profiles are becoming affordable for large-scale studies.

But God knows there are enough vested interests out there who don't want a clear pathology found for CFS. I just hope every last possibility is considered in the XMRV story before it's shut down.

There may be vested interests still who don't want CFS solved, I can imagine some insurance companies, some out-of-date doctors, and a few researchers studying psychological or deconditioning angles. But the major research organizations and govt agencies now acknowledge that CFS is serious and needs a solution. They know we are demanding some equity in research funding. I think many are reading the blogs and forums. And most definitely the virologists and retrovirologists wanted to find answers for us. But they can't study something that they can't find, or that is not even there.

I agree that even if contamination is found and the doubts about XMRV are proven, there still might be some issues related to retroviruses with CFS, such as HERV K18.

 

[alex3619]

By the same measure, the WPI studies only raise the possibility of infection with XMRV. Neither side has conclusively proven its point.

In these early studies, including the WPI study, convenient samples were used. This means the source of control samples was different from the source of patient samples. They were handled and treated differently, etc. In fact Mikovits said at one point they had to test the patient samples many more times than the controls to get the positives to show.

This is why I promote the idea here that we have to let the science run its course before drawing conclusions.

Hi Kurt, I don't disagree with any of your comments, especially the above selected sections. The problem is that there are now many sets of data showing the control vs patient differential, although many remain unpublished. It is highly improbable that a problem with increased handling of patient samples could repeatedly cause contamination with XMRV. The blinded studies may well tell us if there is a problem, and the Singh cadaver biopsies may too. I really wish we had that data now.

Just to reiterate a point I have made many times, if the positive XMRV findings are correct, and XMRV is causal for many diseases, the whole world is in a heap of trouble. I would like XMRV to go away for that reason, but I would also like it to be confirmed too because that could lead to effective treatments.

Bye
Alex

 

[floydguy]

I can imagine some insurance companies, some out-of-date doctors, and a few researchers studying psychological or deconditioning angles. But the major research organizations and govt agencies now acknowledge that CFS is serious and needs a solution. They know we are demanding some equity in research funding. I think many are reading the blogs and forums. And most definitely the virologists and retrovirologists wanted to find answers for us. But they can't study something that they can't find, or that is not even there.

Can you elaborate on the government agencies and research organizations that believe ME is serious and needs a solution? Stanford is the only that I can think of that seems to be doing anything about it institutionally. The CDC? Fauci? NIH? Ha, don't make me laugh. Most of them are responding to pressure to look into it but I have yet to be convinced that any are doing it out of their concern for ME and their desire to make any progress in the field. These folks seem to be quite happy to return to more lucrative and prestigious research in cancer, AIDS or whatever they were studying when they were so rudely "forced" into wasting their time in a backwater like ME Research.

Again don't make laugh about all those virologists doing a two week study on cohorts with everything but ME.

 

[kurt]

Can you elaborate on the government agencies and research organizations that believe ME is serious and needs a solution? Stanford is the only that I can think of that seems to be doing anything about it institutionally. The CDC? Fauci? NIH? Ha, don't make me laugh. Most of them are responding to pressure to look into it but I have yet to be convinced that any are doing it out of their concern for ME and their desire to make any progress in the field. These folks seem to be quite happy to return to more lucrative and prestigious research in cancer, AIDS or whatever they were studying when they were so rudely "forced" into wasting their time in a backwater like ME Research.
Again don't make laugh about all those virologists doing a two week study on cohorts with everything but ME.

Your cynicism is understandable, but I know some people involved and they are working hard on our behalf. The CDC study ran for many months, they constructed multiple tests, ran things many times, even used the WPI PCR test. And they used a very sick CFS cohort with PEM. If XMRV had been found by confirmation testing and it could be found by all the labs, I believe the CDC and NIH would be changing their tune now about CFS and we would start getting more resources for research. But they did not find anything they could easily pin down as a cause for CFS. However, the CDC retrovirology lab did probably jump in because this was about a retrovirus, not because it was about CFS. I won't argue that point.

I did not say they had changed their funding priorities yet, they clearly have not. But these organizations have spent some serious unscheduled funds searching for XMRV, this is a change, even if only the first step towards the major changes needed.

 

[*GG*]

Yes, the hypermutation situation, that is the protein. I don't know if that mechanism has SNPs, we really need some full genomic profile studies in CFS. That in my opinion is where our research dollars should head right now, full profiles are becoming affordable for large-scale studies.



There may be vested interests still who don't want CFS solved, I can imagine some insurance companies, some out-of-date doctors, and a few researchers studying psychological or deconditioning angles. But the major research organizations and govt agencies now acknowledge that CFS is serious and needs a solution. They know we are demanding some equity in research funding. I think many are reading the blogs and forums. And most definitely the virologists and retrovirologists wanted to find answers for us. But they can't study something that they can't find, or that is not even there.

I agree that even if contamination is found and the doubts about XMRV are proven, there still might be some issues related to retroviruses with CFS, such as HERV K18.

"But they can't study something that they can't find, or that is not even there." What are you saying here? XMRV has been found, correct? Are you saying that XMRV does not even exist? Because that's what it sounds like you are saying.

Wasn't XMRV found in aggresive cancers of the prostate back in 2006? Why would someone make that up? You make it sound like no one/or most scientist have no idea what this thing "looks" like. Just because something is not easy to find, does not mean it does not exist! there are many examples of this in the natural world.

GG

PS If a serious researcher really wanted to find XMRV, isn't the WPI "open" to working with most/any interested party?

 

[kurt]

"But they can't study something that they can't find, or that is not even there." What are you saying here? XMRV has been found, correct? Are you saying that XMRV does not even exist? Because that's what it sounds like you are saying.

Wasn't XMRV found in aggresive cancers of the prostate back in 2006? Why would someone make that up? You make it sound like no one/or most scientist have no idea what this thing "looks" like. Just because something is not easy to find, does not mean it does not exist! there are many examples of this in the natural world.

GG

PS If a serious researcher really wanted to find XMRV, isn't the WPI "open" to working with most/any interested party?

I meant that is not even there in the samples being studied, XMRV is real but whether it is being found in CFS samples, or is a test artifact, such as a contaminant somehow getting into samples is being debated by researchers.

Nobody is making any of this up, the science is just not 100% reliable, not on either side of this debate.

To my knowledge WPI is not open to working with just any interested party, most labs are particular about who they work with. Anyway, the CDC ran the WPI assay (PCR) and had different findings. Based on what I have heard, the complaint that nobody has 'replicated' WPI is bogus, if you understand how PCR works. They have all calibrated their tests to the same exact sequence, VP62. And at least one lab has run the same test. Yes, WPI says the culture study is better, but they found XMRV with a standard PCR test, therefore so should everyone else.

 

[Mya Symons]

"This paper has also not shown that XMRV did not arise from recombination in the early 1900s (or any other time) either - the arguments against them are essentially the same as the arguments against the Lombardi et. al. findings, except Lombardi et. al. have way more evidence and XMRV has been found in many labs in controls and patients."

Do you think it would be possible for a virologist familiar with retroviruses to go back over research done using MuLv's in the past (before 1990) to see if a situation could have arose during earlier experiments in which XMRV was accidently created? Or would this be impossible?


"The other confounding factor here is that they are using human prostate cancer cell lines to do the culturing in. The very disease they think is associated with the virus. That creates kind of an awkward situation. Even you find the virus in the culture cells before you add CFS patient blood - you can't really be sure its purely lab artifact because it could have been in those cells because it caused the cancer in the patient the cells came from in the first place. I suppose with 22rv1 they went back and found older cells before they had passed them through mice and found those cells to be negative, so that seems to help the case for that cell line. But I don't know that the same can be said for the other cell lines."

--What a mess. It seems like the only way to finally answer the contamination question is if they start all over with a new cell line (some type of cell that XMRV does not infect) and brand new clean samples from people with Canadian Definition CFIDS (blood and tissue).


"If XMRV had its genesis in the lab, then the greater question is whether or not it has 'escaped' into the human population. There was evidence presented at CROI that is unlikely, as there is more strain diversity in the human cell lines than in the humans who actually tested positive. Based on evolutionary theory, this suggests that the positive tests are likely to result from contamination, as a true infection in the population would by now have far more genetic diversity than the lab cell lines"

Someone mentioned that testosterone was added to the cell line. If XMRV uses androgens to replicate, would it replicate faster in a cell line (where it has unfettered access to testosterone) then it would in the blood of a human? Could that account for the greater diversity in the cell line?


"Other studies have shown that there is a natural human cellular defense mechanism that easily defeats XMRV in a live human"
--Would this apply to those with an RNASEL mutation?

 

[Hope123]

Basic lab stuff is not my area so I don't keep up with all the ups and downs of this but logic made me think the other day, if XMRV contamination is so prevalent because of the cell lines, PCR reagents, etc., why are there a bunch of studies where NO XMRV was found, either in controls or subjects? You would think that they would find SOME positivity either in controls or subjects if contamination was so widespread. Perhaps details of those negative studies need to be looked at again too -- e.g. what cell lines did they used, which reagents, what experiments have been conducted in those labs previously? But then again it's not like science isn't biased right now, right, right? Anyone? [*crickets*]

 

[markmc20001]

It sounds like there are a few different XMRV camps here on pheonix rising. The other competing ME forum, wihtout polling it is pretty safe to say, is generally in concenus that XMRV is real and we need to move foward with treatments now.

where is phoenix rising crowd?

Phoenix rising:
Camp 1:

CDC has poured tons of money into trying to finding XMRV. It is a contaminant camp and probably hasn't escaped to the population camp.

Camp 2:

XMRV is a real virus, can infect human tissue, and waiting for more science to prove or disprove if it is prevalent in general population or in fact just some kind of lab mix-up.

camp 3:

XMRV is real, lets get on with the treatment studies

camp 4: have no clue, waiting for what the concensus comes up with.

Might make a good poll, but don't know how to do that. :0)

 

[Mya Symons]

It sounds like there are a few different XMRV camps here on pheonix rising. The other competing ME forum, wihtout polling it is pretty safe to say, is generally in concenus that XMRV is real and we need to move foward with treatments now.

where is phoenix rising crowd?

Phoenix rising:
Camp 1:

CDC has poured tons of money into trying to finding XMRV. It is a contaminant camp and probably hasn't escaped to the population camp.

Camp 2:

XMRV is a real virus, can infect human tissue, and waiting for more science to prove or disprove if it is prevalent in general population or in fact just some kind of lab mix-up.

camp 3:

XMRV is real, lets get on with the treatment studies

camp 4: have no clue, waiting for what the concensus comes up with.

Might make a good poll, but don't know how to do that. :0)

Going back anf forth between 2 and 3 right now.

 

[*GG*]

It sounds like there are a few different XMRV camps here on pheonix rising. The other competing ME forum, wihtout polling it is pretty safe to say, is generally in concenus that XMRV is real and we need to move foward with treatments now.

where is phoenix rising crowd?

Phoenix rising:
Camp 1:

CDC has poured tons of money into trying to finding XMRV. It is a contaminant camp and probably hasn't escaped to the population camp.

Camp 2:

XMRV is a real virus, can infect human tissue, and waiting for more science to prove or disprove if it is prevalent in general population or in fact just some kind of lab mix-up.

camp 3:

XMRV is real, lets get on with the treatment studies

camp 4: have no clue, waiting for what the concensus comes up with.

Might make a good poll, but don't know how to do that. :0)

I tried starting a poll for you mark, but

Camp1 needs to be shortened from this:

CDC has poured tons of money into trying to finding XMRV. It is a contam. camp and probably hasn't escaped to the pop. camp

and Camp 2:
XMRV is a real, can infect, and wtng for more science to (dis)prove if prevalent in gen. pop. or in fact just some mix-up.

They need to be 100 characters or fewer. What a pain!

GG

PS I did the count in word, but I think this site is counting the spaces?!