XMRV Negative Results -Need for Replication Study

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http://www.cfids.org/cfidslink/2010/010603.asp

CFIDS Association of America

XMRV Negative Results Emphasize

Need for Robust Replication Study

Suzanne D. Vernon, PhD

Scientific Director

A study testing for evidence of XMRV infection in CFS

patients in the United Kingdom has reported

negative results.

This is the first publication following the article in

the top-ranked journal Science from researchers at

the Whittemore Institute, the National

Cancer Institute and Cleveland Clinic that garnered

worldwide attention from the media and scientific

community.

The new report [http://bit.ly/7QwkF5], published

Jan. 6, 2010, in the open access online journal PLoS

ONE, failed to detect XMRV in CFS, but should not be

considered a valid attempt to replicate the findings

described by Lombardi et al., in the Oct. 8, 2009

Science article [http://bit.ly/4HU4j1]

The PLoS ONE paper by Otto Erlwein, Steve Kaye,

Myra O. McClure, Weber, Gillian Wills,

Collier, Simon Wessely and Cleare is

titled, " Failure to Detect the Novel Retrovirus XMRV

in Chronic Fatigue Syndrome. "

The investigators tested peripheral blood DNA from

186 routine clinic attendees who met 1994 (Fukuda)

CFS case definition criteria and were well-

characterized from participation in prior

neuroendocrine and cognitive behavioral therapy

studies.

These 186 CFS patients were reported to be unwell

for a median of four years with high levels of fatigue

and disability.

This team of researchers used a special type of DNA

" xeroxing " called nested polymerase chain reaction

[http://bit.ly/8lv7wb] (PCR) reaction to amplify

specific segments of the XMRV proviral DNA from the

genomic DNA obtained from these 186 CFS subjects.

In essence, they were looking to see if XMRV genetic

material had integrated into human genetic material,

which is a key characteristic of retroviral infection.

The experiment included positive, negative and

contamination controls, but did not test any samples

taken from healthy subjects.

The samples were coded so that the origin of the

DNA was not known to the person conducting the

PCR assays. XMRV was not detected in any of the

186 samples.

Can this study be considered comparable to the

results published by Lombardi et al., in Science?

In short, no.

Both studies included CFS patients defined by the

1994 case definition criteria, but this is where the

comparability ends.

Here are some of the ways the PLoS ONE and

Science methods differ:

* The blood was collected from CFS patients in

different types of blood collection tubes.

* The genomic DNA was extracted and purified

using different techniques.

* The amount of genomic DNA included in the

amplification assay was different.

* Different primer sequences were used that

amplified different regions of the XMRV proviral

DNA.

* The conditions of the PCR amplification assay

were different – from the numbers of cycles, to the

type of polymerase used.

Should these differences affect an

investigator's ability to detect XMRV?

To a microbiologist with experience handling samples

and studying various infectious agents (as I am),

these variances in procedure could make the

difference between detecting XMRV or not.

It very well could be true that XMRV is not present in

the U.K. as Erlwein, et al. suggest in their

discussion, but it is also possible that the technique

used in the PLoS ONE paper was suboptimal due to

the different methods employed, when compared to

the original experiments conducted by Lombardi, et

al.

The U.S. Department of Health and Human Services

Blood XMRV Scientific Research Working Group

[http://bit.ly/8oJ0dt] is conducting a rigorous study

to detect XMRV.

Multiple laboratories will standardize methods to

optimize sensitive detection of XMRV proviral DNA

and viral RNA and then, once methods are

standardized, these same laboratories will test

coded panels of blood samples obtained from healthy

blood donors and CFS patients.

We look forward to the results of this study and urge

that it be completed expeditiously, especially in light

of this report from the U.K.

In the meantime, be prepared to read about more

studies with conflicting findings. Rather than simply

accept or dismiss new information, we will help make

sense of why discrepant results occur.

Perhaps the most important statement in the PLoS

ONE paper is the acknowledgement by this group of

investigators that CFS is an incapacitating organic

disease affecting millions of people worldwide.

Once XMRV detection methods are optimized and

made widely available, we encourage this group of

researchers to take another look at XMRV as a

possible explanation for the organic basis of CFS in

the U.K.

Citations:

Erlwein O, Kaye S, McClure MO, Weber J, Willis G,

Collier D, Wessley S, Cleare A. (2010) Failure to

detect the novel retrovirus XMRV in chronic fatigue

syndrome. PLoS ONE 5(1):e8519.

doi:10.1371/journal.pone.0008519

Lombardi VC, Ruscetti FW, Gupta JD, Pfost MA,

Hagen KS, DL, Ruscetti SK, Bagni RK,

Petrow-Sadowski C, Gold B, Dean M, Silverman RH,

Mikovits JA. Detection of an infectious retrovirus,

XMRV, in blood cells of patients with chronic fatigue

syndrome. Science 8 October 2009. 1179052.

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Suzanne D. Vernon, PhD, earned her doctorate in

virology at the University of Wisconsin at Madison

and worked in public health research on infectious

diseases at the U.S. Centers for Disease Control and

Prevention for 17 years before joining the CFIDS

Association of America's staff as scientific director in

2007.

She has more than 70 peer-reviewed scientific

publications on topics including human

immunodeficiency virus, human papillomavirus,

cervical cancer and chronic fatigue syndrome.

Dr. Vernon has initiated and participated in

numerous international and multidisciplinary research

collaborations and she now leads the CFIDS

Association's research program. The CFIDS

Association of America is the nation's largest

philanthropic supporters of CFS research.