I am so sick of this crap. BUT! A group in China made a really neat Real-Time PCR strategy for detecting XMRV, so Im still gonna write about it :)
Taqman Real-Time PCR is one of my most favoritest things ever. PCR is 'specific', in that we use primers designed to detect specific sequences in a sample. But its not really all that specific. The primers you use dont need to match up perfectly to the sequence you are interested in. Scientists capitalize on this to introduce mutations, or to fish around for all sequences that are 'kinda like' what they are interested in (like how many different places has KoRV inserted into a koalas genome), etc.
This is a problem when you really are looking for something specific, though. The gels in the last XMRV paper look like shit because of non-specific primer binding and amplification.
How do you *know* what you are seeing is what you are looking for? Taqman probes. You add a third primer to your PCR reaction that should recognize sequence in between your two original primers. When DNA polymerase hits the third primer, its like a train hitting a Honda parked on some railroad tracks. The third primer gets chopped up, releasing a fluorescent reporter that a computer reads. If you have non-specific primer binding, but the probe doesnt recognize sequence in between your primers, then the computer doesnt see it. All those non-specific bands in those crappy gels? They disappear.
You can add lots of primers with different colored probes in one reaction (called multiplexing) to look for several things in the exact same sample (I look for two different reporter genes, HIV-1, and beta-globin in one Real-Time PCR reaction).
And, Real-Time is much more sensitive than regular PCR. You know, you see those bands in gels? For the human eye to see even the faintest band, you have to have like a bazillion copies of sequence amplified. For Real-Time? Its a computer looking for those little fluorescent blips. It can literally see one copy amplified. In this paper, they got down to being able to detect 10 copies of XMRV in one milliliter of sample. To put this into perspective, I regularly generate HIV-1 stocks at 1,000,000 IU/ml. Their method is sensitive.
In this paper, they used primer-probe sets targeted towards a rather conserved region of gag. If you BLAST their primer-probes, you not only get XMRV as top hits, but also the 'human MLV' sequences entered by Alters group. While they only talk about XMRV in this paper, they also exclude the sequences Alter found.
They also worked out a neat way to make sure there wasnt anything wrong with their DNA-- a competitive internal control. Normally, we use beta-globin or GAPDH or actin or some kind of normal cellular gene as a control in PCR. But those are non-competitive. Primers have different properties and different specificities-- your control might be fine, while your experimental is f-ed up. So they created a plasmid identical to XMRV, with an altered probe binding site to spike into patient samples. So, the primers would still amplify the same sequence on the plasmid as in XMRV, but if the XMRV probe couldnt bind, the computer wouldnt see this internal competitive control. If they added an appropriate probe (with a different color), then they could 'see' the competitive internal control. If they got any XMRV+ samples, they could take away the XMRV probe and add the competitive control probe and make sure the signal disappeared. If the competitive internal control didnt work, then they know their PCR was fucked up, and they wouldnt detect XMRV even if it was there. I have never seen this used, and I think its wonderfully clever.
They also used appropriate groups-- 1) CFS patients, 2) healthy blood donors, 3) sick but should have no connection to XMRV/CFS patients (HIV, HTLV, hepatitis patients). We were literally just talking about the importance of group #3 in the last post.
Theres only so much you can say about a negative paper, but I wanted to give this group props for making a pretty ingenious system for detecting and monitoring XMRV in a quantitiatve manner. As Ive said before, even assuming XMRV exists and is pathogenic, it is pointless to prescribe antiretrovirals to 'infected' patients until we have a way to quantify their efficacy.
- Log in to post comments
More like this
You should contact Dr. Joseph Brewer, Dr. Jose Montoya , Dr Joe Burrascano, Dr. Daniel Peterson, Dr. Paul Cheney and Dr David Belll Dr. Ila Singh them what they think of viruses in ME/ CFS.
WPI just published again in Aids Reviews. Maybe they are right. I'm giving them the benefit of the doubt.
This makes me think of a paper with the title "Failure to detect the Loch Ness Monster in Loch Ness"; where the methods section includes "We used sonar. And radar. And baited hooks. And nets. And we bailed out the Loch with a teacup until it was completely empty and poured every teacup through a seive. We did not see Nessie."
But then.... we can still say that the negative outcome is caused by regional differences. China is very far away.
i have no idea what you're saying, but i love your blogs. I'm sick and I want to get better, but i dont want to drink kool aid. you're refreshing, thanks
Judi, short version, there is a Nobel price for the group that finds a cause for CFS, similar to the people that found HIV to cause AIDS. Some people claim the equivalent in CFS is XMRV, but it's not panning out when looked at rigorously. And this paper describes testing done VERY rigorously.
Jacqueline-- That was my initial conclusion when the negative papers popped up.
XMRV/human-MLV/magic-eye-MLV could be infecting people in the US, and might be biased in populations that are otherwise immunocompromised. That is not evidence that the virus is causing the underlying disease, but it could be perpetuating some symptoms of said disease in some people. Or it could be causing specific cases of said disease, epidemiologically recognizable as 'outbreaks', but might not be The Cause for The Disease in general.
But thats not what Judy/WPI/Alter are saying. They are saying that a species of virus is The Cause of The Disease and they can find it in all of their samples, from all over the world.
No one can replicate those results, including each other, including labs using better technology and better controls.
Judi-- Ask questions if Ive said something you dont understand! Thats what Im here for! :)
@erv I was being sarcastic ;)
(although I initially also thought regional differences might be a reason, but I had to dismiss this, when Judy claimed to find XMRV in the negative Dutch & UK samples)
I bet Judy can find something in those Chinese samples too. With 100% accuracy...
Meanwhile...
http://www.mountainx.com/news/2010/091510local-family-feels-vindicated-…
"The one-page letter dated Sept. 1, 2010, contained the following statement:
"I wanted to inform you that your son Ryan's tests indicated that he has positive [sic] evidence of XMRV in his blood sample drawn by PSI several months ago. As you know, we are just at the beginning of understanding what this means and what the implication may be for Ryan and your family."
The letter, addressed to Lisa Baldwin, was signed by Judy Mikovits, director of research for the Whittemore Peterson Institute for Neuro-Immune Disease"
And from the comments section of the story:
"The letter cited in this story noted the following:
'We have been exploring antiretroviral treatment strategies in collaboration with several experienced HIV physicians who have used the ICD9-Code 079.59 (specific retroviral infection).'"
In other words, they are prescribing people ARVs and using this code to get reimbursement.
The XMRV workshop doesn't sound like it helped much:
http://www.sciencemag.org/cgi/content/short/329/5998/1454
"the field remains mired in "a zone of chaos," organizers concluded."
From the Q&A session at the end of the XMRV conference it sounds like the blood working group looking at XMRV figured out what is causing the failure to detect a couple of weeks ago. Apparently collection and handling of the samples is key. I can't remember in which clip they talk about it.
http://www.youtube.com/user/5qurple#p/c/C48EFC7D6D7F217A/3/VC1yPg184bw
Ruscetti was confident enough at the conference that he stated that he isn't going to discuss the detection issue any more and is moving on to focusing on pathology.
Honestly, ERV I think you are beating a dead horse on this one.
Or maybe you are setting yourself up to be a XMRV denier just in case you find God or something? :-)
There is no published evidence to support that kind of confidence (arrogance).
In the absence of that evidence, making such claims is kookish, not being skeptical of said claims.
This is getting silly. Can we not just do some whole-genome sequences on these patients and be done with? They're knocking them out for $10k or less these days.
Seriously, Impish? Mikovits also talked with great confidence before the conference, but the results were underwhelming to say the least. Everyone has been confident *their* results are correct, so focusing on the confidence of Ruscetti feels a bit selective.
Also, that blood working group you mention has not produced flattering results for the WPI in their first fase of blinded testing. First, it is a mystery why the WPI decided not to use their (superior if their results are correct) *Science* PCR assay to detect XMRV in the samples that were sent. The other labs (e.g. Switzer and Alter) did use the same assay they used in their papers. Second, although all labs were able to detect 14 or more spiked xmrv cells per ml of whole blood, WPI's assay did not perform well beyond that.
To compare, the CDC (Switzer) detected 89% (8 out of 9) of samples with little (< 4.5 cells/ml) infection and produced 0% (0 out of 6) false positives. WPI detected 44% out of the same samples and produced 1 (17%) false postive.
Mikovits explained this by saying they didn't use the Science PCR assay. WTF?
It's (still) very well possible that XMRV and CFS are connected, but this confidence you're speaking of means nothing.
How the fuck is that legal? How the FUCK is that LEGAL?? How the fuck can Judy be so stupid as to sign her name to that??
The 'test' sold by the WPI has NOT been approved by the FDA.
Even assuming that XMRV is 100% real and pathogenic, the test has not been approved by any government regulatory agency. At best, its a lab hack. We do shit all the time, in the lab, for the detection of HIV/MMTV/MLV that you would never use in a clinic. Every reagent we use in the lab says on product inserts and right on the damn box "NOT FOR CLINICAL USE. RESEARCH PURPOSES ONLY."
If this turns out to be a bust, anyone labeled 'XMRV POSITIVE' could sue the WPI and Judys asses off for mental distress, medical bills, etc etc etc. Insurance companies could sue for fraud... christ.
Fucking idiots, I swear to fucking god. Even if they are 'right', they are fucking idiots.
Don't be silly, Stephen.
Didn't you see that film with the robot that walked through bars? How do you know that Nessie isn't capable of dissolving and reprecipitating in loch water? Eh? Eh? How?
I submit to your honor that right up to the point where he entered renal failure from his parents illegal administration of AZT little Ryan's parents repeatedly described his ME/CFS/WTFBBQ as being worse than triple aids with a pancreatic cancer back flip.
Shall we penalize Dr. Mikovits because she let a beam of light shine into their dark and hopeless herfhurfdurfedydurf....I think not.
Nothing further.
The real cause of CFS is not enough mitochondria. One of the most common (and unacceptable) side effects of antiretrovirals is mitochondria depletion. It is mitochondria depletion that causes the liver failure and kidney failure and brain failure associated with antiretroviral treatments. Those are three tissue compartments that have a lot of mitochondria.
http://www.molecularneurodegeneration.com/content/5/1/27
It causes muscle failure too. Mitochondrial myopathy has been associated with antiretrovirals for 20 years. There was a paper on it in NEJM in 1990.
Mitochondrial Myopathy Caused by Long-Term Zidovudine Therapy
Antiretrovirals would make CFS worse, not better. There might be a placebo effect, but if they start to feel absolutely great, it is probably near death metabolic stress. At that point even more nitric oxide probably won't help ;) (but before it gets that bad....?)
Judy Mikovits, PhD didn't prescribe anything, she isn't that kind of doctor. It would be the MDs who prescribed the antiretrovirals that would be gone after. They have malpractice insurance too, but their insurance company probably has a disclaimer for illegal stuff and experimenting on people without IRB approval isn't going to fly.
Maybe Ryan really is ill because of the psycho-social strains created by his parents and the WPI's lab results are going to further worsen his mental illness and make it harder for social services to intervene and get him the help he needs.
Maybe Ryan is ill because of XMRV/whatever and the WPI's test results are going to be able to free him and his family of the burden of being treated as if it is their actions making him ill, along with the fears and self-doubt this will create.
It may be idiotic of the WPI to open themselves up to such a risk of lawsuits in this way, but if they think they're right and that a lot of patients are currently being abused by a system which can assume psychological disturbance whenever there's a diagnosis of CFS, there are understandable and noble reasons for wanting to intervene early.
I don't know what I think they should do in such a situation. My first instinct was that it was irresponsible of them - but it's interesting thinking about it while the science is so uncertain. Interesting and a bit confusing - I think I'm going to sleep on this.
Actually, just testing people and telling them the result would require IRB approval. It is research. There is no known therapy, it is pure research. Telling someone their XMRV status (with gigantic and unknown error bars) doesn't fall within the treatment exemption because there is no treatment.
I don't think that WPI has a clue what is going on. I think it is wanabe fools rushing in where experienced and knowledgeable grad students (and everyone who has even half a clue) fear to tread. Maybe WPI has enough political connections that they can cover this up. Provided no one dies from antiretroviral induced liver, or kidney, or brain, or muscle failure.
Any idea what this could be? http://www.theepochtimes.com/n2/content/view/37509/
I am unclear what makes this an 'AIDS-like' disease. Nothing about those symptoms resembles HIV/AIDS. Those symptoms are far too broad for me to suspect any virus, including a retrovirus. I am not a physician, but something that could cause those broad symptoms is poisoning-- either chemical or radiation.
Im sure the answer isnt that simple, though, because Im sure Im not more clever the physicians seeing these patients.
But nowhere in that article do they say "loss of B-cells"/"loss of CD8+ t-cells"/"loss of any immune cell or any sign of measurable immunodeficiency of any kind". Those look to be a collection of symptoms that scared patients are self-diagnosing as 'AIDS-like' (wtf is 'lymphatic pain'? and your fat hurts? wat?). Actually, all of that article looks like scared patients making stuff up, the best they can.
But that doesnt mean its a virus. Viruses are just useful ideas for scared sick people because they are alien. Viruses are easy scapegoats in pop culture, which is one of the reasons I started ERV.
Viruses are not magic. They dont make your 'skin feel like fiber', but you appear normal and healthy to physicians. Either there is a virus with a pathology and a disease, or theres not.
ERV states "I am unclear what makes this an 'AIDS-like' disease. "
I would refer you to the work of Dr Klimas, an HIV and M.E doctor, she herself states the similar findings in HIV carriers and M.E patients.
Also I have come across a large number of M.E patients with lower white cell counts than healthy controls, their white cell counts following a similar pattern of HIV carriers, among a number of patients I have spoken to many have been offered HIV tests by their doctors becuase their blood work basically is similar as HIV carriers, which does beg the question if it isnt HIV what the heck is suppressing their white cells.
EDIT, I have just realised the comment is referring to the chinese article in the posting above, which I didnt realise, but my comments re M.E and HIV still stand. :-)
"I would refer you to the work of Dr Klimas, an HIV and M.E doctor...."
I would refer you to the fact that Nancy Klimas has established a private clinic and is charging CFS/ME patients $1,400* for their initial consult and test, having hitched her star to the WPI wagon within 72 hours of their initial publication.
This is the same Nancy Klimas who stated, in the NYT, she would much rather have HIV than CFS because her HIV patients are "hale and hearty".
I really am disinclined to give a shit about the opinion or observations of an HIV researcher so myopic she dismisses the lives of the 22 million people in Sub-Saharan Africa who can't afford her anti-retrovirals.
*the examining rooms supposedly have massage loungers.
I have a question that maybe you can answer. There is a doctor claiming that the nested PCR and the substance used by Whittmore Peterson to grow the viruses (sorry I cannot remember the name of this substance) is contaminated with MuLv's. She claims that they store these things in the same area they keep and work with the lab mice. Do you think it is possible that the positive results that Whittmore Peterson claim they are getting are really caused by contamination of MuLv's already in the nested PCR and this substance used to grow the viruses?
"I am unclear what makes this an 'AIDS-like' disease. Nothing about those symptoms resembles HIV/AIDS."
Are you seriously so lazy you can't even bother to do a quick search to look at the literally dozens of published papers that outline the immune disfunctions that occur in CFS patients.
CFS is a shitty shitty disease. My sister has it and has had it since 1985. Her immune system is so fucked up that she gets each every flu and cold that comes near her. The latest thing was her arm and leg swelling up with an infection that her body with the help of antibiotics was barely able to fight off.
If you want to pretend to be an expert on something and have a blog about viruses you might do at least a trival amount of research before posting something.
You realize I was commenting on the article linked to by Ernie on an 'AIDS-like illness' in China, you emotional retard?
prometheus your comment is so full of innacuracy and bilious venom I dont know where to start, Klimas was talking about HIV carriers who can actually lead relatively normal lives given the right meds, YES of course its a total tragedy about the african carriers but that is a totally different issue, klimas is talking about the USA, hence in the USA she would rather have HIV than CFS, remember AIDS is the result of HIV but the symptoms are different, also Klimas is a university based researcher, that work is separate to her clinic, if she wishes to have a clinic then so what? if she could give her time free maybe she would but the usa healthcare system isnt set up like that.
if you want to be taken seriously prometheus then check facts and dont make wild accusations about immunologists who seem far more qualified on these matters than yourself
Okay, anybody who thinks "immune disfunction" means "AIDS-like" gets to shut up while grown-ups are talking.
Have you read the latest issue of science, ERV? There was this tiny snippet of information about the second fase of testing by the blood working group, for which Judy Mikovits collected positive samples from the Science study:
"The group has also tested four WPI samples from CFS patients but isnât ready to discuss the results because âweâre still confused by them,â says Coffin, who is part of the working group."
Mmmmmm.....
jakob@#27
"prometheus your comment is so full of innacuracy and bilious venom.."
As am I. So very full. Like a Gila Monster.
Also, I can spell inaccuracy and use a shift key.
Moron.
P.S. Nancy Klimas stands to administrate billions if Mikovits is vindicated. This will be as an administrator for the Veteran's Administration, not as a lab director for Miller of Miami Health. If Mikovits is wrong Klimas has Miami U tenure, a private cfs/me clinic/spa and can take early fed retirement at about 85% next year.
I indicated she was biased, I didn't say she was stupid or even wrong.
lol prometheus, resorting to pseudo intellectual insults is hardly a way of getting yourself taken seriously. "administrate billions" lol that really DID make me laugh out loud, you seem to be quite paranoid and think everyones on the make here.
p.s EVERYONE is biased, in the WHOLE world, its human nature to stand by what you beleive in, YOU are biased, I am biased, ERV is biased, get over it, you are just using selective bias to try and add weight to your arguement. What a scientist would call research bias. I if everyone dropped their studies as soon as another scientist disagreed with them then no science would get done.
I dont even care what you think of XMRV etc what I do care about is good science.
Well then, how much budget will the chief of treatment protocols for "XMRV as culprit" control on behalf of the Department of Veterans Affairs?
Keep in mind the VA spent 42 million on phone calls last year.
The fed numbers for HIV/Aids are up for 2011.
27.2 billion.
There is a slight difference between "I am biased towards dark blue versus light blue." and "I am biased towards enormous power and saintly posterity versus retired obscurity."
Maybe, like Klimas, you only understand degrees of bias or human suffering when it serves your ambition.
While I am here.... calling you a moron isn't an intellectual insult or a pseudo-intellectual insult. It is just a garden variety derogation. Res ipsa loquitor, you are still a moron.
lol prometheus you are the one resorting to pathetic insults, so I will rise above those and try and engage with you.
what you fail to see is that there are always two sides to everything, so in your world klimas is just in it to make some cash (your opinion and bias), but then you fail to see that there are just as many other people who are desparate for xmrv to fail becuase then THEY wont be able to cash in indeed there already ARE people making shed loads of cash out of desparate patients.
Any disease that is not fully understood is first preyed upon by sharks and charlatans, its happened all throughout history. Then once the science works out whats going on then the people get treated and stop getting exploited. History is littered with such scenarios.
re Klimas I see nothing that she has done that would worry me. She seems genuinely interested in science and especially the science of HIV and XMRV, M.E etc, can see much wrong with that!
so hence my statement about, all I am interested in is GOOD science.
how do we know if the subjects in china were really "CFS patients?" do we even know what CFS is since there are several definitions? people who have all sorts of other conditions (lyme disease, MS, major depression, Lariam poisoning, mold allergies...) are often diagnosed with CFS.
No one has CFS except for people who REALLY have CFS.