New Technology finds XMRV – Dr. Lapp sounds quite excited in his recent newsletter. Its not often you read Dr. Lapp reporting “that (the) information relayed this past week to us will change the field of medicine forever”..You don’t hear that very often!
He reported that Dr. Urnovitz of Chronix Biomedical Labs. Chronix has developed an incredibly quick and inexpensive way of mapping the entire genome which he reported will be available soon. When cells die they spill their guts into the bloodstream…its these sequences that Dr. Urnovits technology is looking for. When they looked in the blood of some CFS patients they were able to pick out ‘chimeras’ made up of XMRV genes (but oddly missing their LTR regions). (Check out the full description here“Urnovitz revealed that his research group is able to map genomes at a very rapid pace. He expects that in the near future, Chronix will be able to map your entire genome in under six hours and for probably less than a $100 fee. This is StarTrek medicine!
Urnovitz went on to explain that when apoptosis occurs, chimeras are spilled into the blood stream and can be extracted easily by his laboratory. When his lab examined the genomes of persons with CFS they found chimeras made up of XMRV genes (but oddly missing their LTR regions). “
On March 3rd Hemispherx and Chronix teamed together to file a joint patent for a blood test to diagnose CFS using these techniques.
It’s definitely sounds like an interesting approach; they are, after all, looking at dying and damaged cells – which would seem to be a good thing to focus on in disease. They are using this technology with a few other disorders but haven’t published any papers using their technology.
Dr. Urnovitz has run several labs over the past 30 years and has published 3 papers over that time that I could find. This is really new technology….and it remains to be seen how it will be accepted. I wouldn’t expect any dramatic turnarounds in the medical field regarding XMRV from this and I suspect that the key studies on XMRV will be finished long before this research really comes into play but it is some positive news for XMRV after a couple of weeks of mostly bad news.
Lo/Alter on XMRV – http://videocast.nih.gov/Summary.asp?File=16477 Dr. Alter started off his presentation on Demystifying Medicine stating that this course might be better called “Mystifying Medicine” and said it would probably move the audience from confusion to utter miscomprehension and that there are many unanswered questions.
Speaking in a very clear and methodical manner, he noted that the pathogens in question, XMRV and the polytropic MLV’s, have “not been accepted by most of the scientific community” and that chronic fatigue syndrome does not have an established etiology.
He then went through the history which began only in 2006 with the Urisman/Silverman discovery and XMRV’s primitive nature relative to other retroviruses and the rather sordid history of MLV’s – causing neurological diseases and cancers in mice.
Why CFS? He noted the outbreaks and the tendency of the disease to be triggered by an infection and the possibility of an underlying immune deficiency and the strong association with CFS (PCR, antibody, culture) demonstrated in the Lombardi et al paper.
He said that ‘made a nice case!” and then went over the series of negative studies that came out and then came the Lo study….looking at one set of CFS patient and a set of controls from a blood bank, with its finding of MLV gag sequences in 86% of CFS patients and 6% of normal donors. He called this ‘sort of confirmatory’ but not ‘fully confirmatory’ because Dr. Lo had not found had not found XMRV but a ‘closely related’ polytropic murine leukemia virus (MLV).
Then at the XMRV Symposium he noted that Dr. Mikovits found that 48% of UK patients tested were positive for XMRV and 63% were positive using antibody. Dr. Hanson found 55% of samples (from Dr. Bell) were positive for the same pMLV’s Lo/Alter had found and some new news, at least to me, that Dr. Ruscetti had found a high percentage of patients were positive using antibodies.
Next came the five articles in Retrovirology suggesting XMRV/pMLV’s were the result contamination then he turned it over to Dr. Gill who then passed it to Dr. Lo.
Dr. Lo – started off by stating that he’s been a pathologist for almost 25 years. His CFS samples had been stored for many years after his failed attempt to find mycoplasma’s in CFS. They had two sets of blood samples, some of their own and some sent by Dr. Komaroff 86% of which tested positive for the MLV gag genes.
They used the primer designed from the Science paper and during the first round they found that almost 40% of the samples produced a ‘product’ that was slightly bigger (by 15 nucleotides) than in the first paper. (Nested PCR is done in two the rounds; the first round is done to focus the PCR – the second round then zero’s on that focused area. For the researchers unable to find any evidence of XMRV over two rounds the ability of Dr. Lo to find so much after the first round was surprising). The second round found that >80% tested positive. They then cloned and sequenced all the bands. They also found these sequences using RT PCR in lower numbers.
The Lo Findings and XMRV – They found, though, that the sequences they found lacked a genetic sequence deletion that was originally considered a distinguishing factor of XMRV. A comparison of the first 184 base pairs indicated that their sequences were ‘much more’ closely related to the p MLV’s than to XMRV and were ‘much more different’ from the reported XMRV sequences in CFS or prostate cancer.
A genetic analysis indicated that the sequences they found “could not” be grouped in the same class as the XMRV sequences. An amino acid analysis found the same thing; the sequences they found were more closely related to p MLV’s than to XMRV but it was still ‘pretty close’ to XMRV – if you look at just the amino acid sequences. An analysis of the envelope gene found the same; it was ‘very distant’ from the XMRV sequence and much more closely related to p MLV’s.
Contamination – He noted that most laboratories used ecotropic MLV’s and the sequences they found were from p MLV’s – which are not usually used in laboratories. The big question was the contamination by mouse DNA since the mouse genome contains many closely related endogenous sequences.
Dr. Lo did not rule out contamination was that what we need to do is develop a very highly sensitive assay that can find mouse DNA. They chose mtDNA assay and he noted that each cell contains many mitochondria making it a very sensitive assay. One part of the mouse genome is very different in humans and mice and that is what they looked at. First they spiked the sample with mouse DNA (just as researchers spike samples with XRMV DNA) and then looked for it. They found no evidence of contamination in their samples. (He did not mention anything about the IAP test).
He said they knew their findings would be controversial. He asked why all the different findings? And went through all the reasons…but the one that stuck out for him was ‘variation in the PCR protocol’ and that even though all the labs say they are doing the same assays there are slight differences…in annealing temperatures, magnesium concentrations, etc. and they don’t know if those differences make a difference or not.
The problem is the very low copy numbers in the blood give little margin for error; given that he believes slight differences could tell the tale.
IAP vs mtDNA – he went over the Retrovirology paper which appeared to find XMRV sequences but which an IAP test indicated was due to mouse contamination. They also found that mtDNA testing did not pick up some of the contamination. He thought there was a miscalculation in the study but ‘more importantly’ he noted that they stated that their assay was more sensitive than the mtDNA assay but he disagreed with that.
He also noted that there are different types of mtDNA assays and they were confident in their assay.
He felt that their finding was consistent with the WPI results and then noted a major difference; their MLV sequences showed a good deal variability while the larger WPI study found little variability.
Dr. Lo reported that they sent some positive samples to the CDC lab – which came up negative and that the CDC sent a negative to Dr. Lo’s lab – where it came up positive. The negative sample was notable because the CDC has repeatedly tested it and it came up negative every time yet every time Dr. Lo tested it – it came up positive.
Dr. Alter – came back on and emphasized the viral like nature of the disease but also noted that ‘we don’t know if its a virus and we certainly don’t know if its XMRV.’ and then rattled off several important questions; is CFS a ‘disease’?..If it is is it caused by a virus and if so which one?
On their part they need to figure out if the sequences they’ve found come from viruses or are due to contamination? He suggested that the patients could have been treated with something that introduced the sequences. He went over their own study and how good they thought it was. He felt it was very important that the WPI was able to grow the virus and find antibodies to it consistently.
On their side they’ve had trouble finding the antibodies and are working with the NCI on that. (Dr. Ruscetti has reportedly found them in Dr. Alter’s samples). He felt the most important study was Dr. Lipkin’s study looking at viral onset, Canadian Criteria identified patients (apparently not positives from the WPI) which will be split up into triplicates and then sent out to various labs including the WPI and Dr. Lo’s lab. They will use both the mtDNA and IAP tests to search for contamination.
If the labs cannot find XMRV in those patients then contamination must be considered; if, on the other hand, they find XMRV in the CFS patients the finding will be confirmed and contamination will be ruled out. We should know in the next six months and he said you can believe what you want to believe until then. With a wry smile he said this has a been a very ‘fatiguing’ experience and he was worn out by it.