Friday, February 3, 2012

A request for clarification regarding the 33 XMRV PCR negative patient samples in Lombardi et al. 2009

[Update 2013-09-01] I am no longer expecting an answer. For me, there is no benign explanation, the only plausible explanation for me is the the work by Mikovits, Lombardi and Ruscetti is the result of deliberate fraud – I am not going to waste any more time on this fraud. And BTW, the idiots believing in the lies and BS spread by Mikovits should go and fuck themselves. [End of 2013-09-01 update]

Prompted by my previous post: Does Table 4 lie?

I need to write something about myself first – apologizes for that. If you want to skip that, jump right down to the first headline (Lombardi et al. 2009) below.

It was sometime around February 2011 that I learned of CFS and subsequently of XMRV.

My symptoms were getting worse since 2010. Since the end of 2010 I have been going through the medical information I found on the internet. I was getting desperate. I found all kind of things like "Adrenal Fatigue" and "Chlamydia Pneumoniae". My (new) doctor wanted to shoehorn me into a "Burnout Syndrome" diagnosis. When I found CFS on the internet, I initially rejected Chronic Fatigue Syndrome because it is easy to mistranslate Fatigue into Tiredness in German and I thought to myself: I am not tired!

So in early 2011, after looking more carefully into CFS, after seeing that some of my symptoms might be CFS symptoms and realizing even though my illness was clearly gradual onset (and how some things that I though were "normal" for me, were actually anything but normal), I realized I might actually have CFS.

That was the time I first saw online video of a lecture by Dr. Mikovits. And her talk persuaded me. Especially one part: After the initial CFS/XMRV study (Lombardi et al. 2009) they went back and took the all the 34 patient samples that were negative for XMRVby PCR, and retested these samles with the other methods they had. And Dr. Mikovits reported that they found evidence for XMRV in 31 of the 33 PCR negative patient samples. So this raised the evidence to 99 out of 101 patient samples.

And I thought: "This must be it. If Dr. Mikovits can find evidence for XMRV so easily in practically all patients, then it must be XMRV." I thought this was a slam dunk.

As probably most of you know, there were some researchers who disagreed with these findings. Well, I read Osler's Web at that time, so dissenting researcher did not bother me too much – let them criticize. After all, Dr. Mikovits found XMRV in 99 out of 101 patients!

I was contemplating getting an XMRV test. The cost of a XMRV test would have blow my budget for medical issues, so I choose to wait. Instead I did other blood tests, as I had a long list of things I wanted  tested…

Over the last months I started to look more carefully into the XMRV research and the one point I will focus in this post is the 34 PCR negative patients – as this was the evidence that convinced me XMRV is clearly associated with CFS.

If you find something that I have written here below that is not supported by the facts as reported by Dr. Lombardi or Dr. Mikovits: Please point me to the correct information!

(all highlights are mine)

Lombardi et al. 2009:
33 PCR negative patient samples
68 of 101 with XMRV gag sequence

This study was submitted May 7th 2009, accepted August 31st 2009 and published online October 8th 2009.

It found:
Of the 101 CFS samples analyzed, 68 (67%) contained XMRV gag sequence.
(The PDF is available e.g. here)

So with 68 out of 101 reported positive, this makes 33 CFS samples negative in May 2009.

October 2009:
33 PCR negative patient samples retested
99 of 101 with evidence of XMRV infection

In the CFSAC October 2009 meeting Dr. Peterson of the WPI reported that the 33 PCR negative samples were retested with all methods and 31 were found with evidence of XMRV infection:
Evidence for the presence of XMRV in 33 PCR Negative US CFS Patients
19/33 Antibodies in the plasma
30/33 Transmissable virus in the plasma
10/33 Protein expression in Decitibine (5Aza2DC) treated PBMC

Thus, since the submission to Science we determined 99 of 101 US patients show evidence of XMRV infection
This slide of was used by Dr. Mikovits in her talks and is present for the talk on January 22nd 2010.

(Slides available e.g. here and here)

So in October 2009 we have "evidence of XMRV infection in 99 of 101 US patients.

Mikovits et al. 2010:
34 PCR negative patient samples
XMRV detected in more than 75%

In the study Mikovits et al. 2010 (Called "Addendum to Lombardi et al. 2009" by some), which was submitted Feburary 26th 2010, revised May 20th 2010 and accepted May 24th 2010, the following was reported:
In October 2009, we reported the first direct isolation of infectious xenotropic murine leukemia virus-related virus (XMRV). In that study, we used a combination of biological amplification and molecular enhancement techniques to detect XMRV in more than 75% of 101 patients with chronic fatigue syndrome (CFS).
(PDF available here)

Why has the detection gone down from 99 of 101 in October 2009 down to 75% of 101 in May 2010? Is there an explanation somewhere?

Furthermore this addendum states:
Of the 34 patients whose PBMCs were negative for XMRV by DNA or cDNA PCR, 17 were positive for infectious virus when co-cultured with the LNCaP indicator cell line, as XMRV gag and env PCR products were detected in the cell line following their infection with XMRV from the patient PBMCs (Table 2).
The discrepancy between 33 and 34 is fretful, but probably only a typo that wasn't catched.

Mikovits et al. 2010, Table 4:
Only 93 patient samples reported
20 PCR negative patient samples (up to 28 negative if including all missing)
73 patient samples are positive (up to 81 negative if including all missing)
Seemingly several inconsistencies

Now in table 4 of the "Addendum" it gets really weird.

(You can find the table in the PDF, a copy of the table is available here).

First of all only 93 samples are reported, 8 are missing. Ok, this might be some benign mistake of accidental omission – but is annoying as heck if we should take this as evidence that a retrovirus is circulating in the population.

None of the controls are reported. Not even the positive controls. Why? What about the 8 out of 218 (3.7%) of healthy controls infected with XMRV? Why aren't they reported? Were more controls tested by other methods? There are several questions that arise from the controls, that would probably bust the scope of this topic.

73 of the 93 are positive by PCR. Lombardi 2009 reported 68 PCR positive. How did 5 samples turn positive?

20 of the 93 are negative by PCR. If we assume all 8 missing patient samples are negative we now have 28 PCR negative samples at max. This is clearly at odds with the quote above ("34 patients whose PBMCs were negative for XMRV by DNA or cDNA PCR"). What is going on here?

But wait, it gets better!

The following patients were negative by PCR, but not tested (nt) by other methods.
1117 - - nt nt nt
1120 - - nt nt nt
1134 - - nt nt nt
1145 - - nt nt nt
1148 - - nt nt nt
1154 - - nt nt nt
1155 - - nt nt nt
1164 - - nt nt nt
1167 - - nt nt nt
1170 - - nt nt nt
1175 - - nt nt nt
1176 - - nt nt nt
Now what the heck is going on here?

But wait, it gets better!

Table 2 (that is mentioned above to show the 34 PCR negative patient samples) show first of all the results only for 11 patient samples:
But wait, it gets better!

If we take the patient number from table 2 and look them up in table 4, this is what we find:
1103 + +
1111 + +
1132 + +
1150 + +
1169 + -
1172 + +
1173 + +
1186 + +
1189 + +
1199 ? ?
1220 + -
1221 + - 
All samples reported in table 2 (some of the "34 PCR negative samples") are PCR positive in table 4 (with the patient sample 1199 missing).

This simply does not add up.

Is there some benign explanation I am missing for this?

I would dearly like to have an explanation for this.

(Posted in the PhoenixRising forumsmy followup was moderated and has not yet appeared)

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