A riff on the Racaniello article
http://www.virology.ws/2011/05/04/ila-singh-finds-no-xmrv-in-patients-with-chronic-fatigue-syndrome/
From Dr Racaniello - after noting all the problems with the earlier studies, Dr. Racaniello called the Singh study the 'most comprehensive study to date' and, importantly, that it took into account a number of potentially confounding factors that could have contributed to past negative results (differences in patient characterization, geographic locations, clinical samples used, and methods). That was its purpose all along. The killer here is the inclusion of 'methods' since this implies that Dr. Racaniello believes this study did all the methods right.
Since the first association of the retrovirus XMRV with chronic fatigue syndrome in 2009 in the US, subsequent studies have failed to detect evidence of infection in patients from the US, Europe, and China. These studies were potentially compromised by a number of factors, such as differences in patient characterization, geographic locations, clinical samples used, and methods used to detect the virus. These and other potentially confounding conditions have been addressed in the most comprehensive study to date on the association of XMRV with CFS.
Study parameters
To address these issues, the authors collected blood from 105 CFS patients and 200 healthy volunteers in the Salt Lake City area. One hundred of the patients fulfilled both the CDC-Fukuda and the Canadian consensus criteria for diagnosis of ME/CFS. The patients were selected from a clinic that specializes in the diagnosis and management of CFS and fibromyalgia.
The clinic, of course, was Dr. Bateman's clinic - a clinician who knows how to characterize CFS patients.
They looked for XMRV using f
our different PCR assays and ELISA antibody and they tried to grow the virus in cell cultures; the same approaches, Dr. Racaniello reported, used by Lombardi et. al in the original paper.
New blood samples were also collected (by a third party) from 14 patients from the original study by Lombardi et al. The samples were blinded for subsequent study. Detection of viral nucleic acids was done using four different PCR assays. Anti-XMRV antibodies in patient sera were detected by ELISA. Finally, virus growth from clinical specimens was attempted in cell culture. The authors used the multiple experimental approaches reported by Lombardi and colleagues
.
As was noted many moons ago - the pol region is the most specific for XMRV since that region rarely changes in viruses. They also looked for the gag and env regions (as the WPI did). As with many of the other studies, everybody was negative via PCR and antibodies - patients and controls.
PCR for viral nucleic acids. Four different quantitative PCR assays were developed that detect different regions of the viral genome. The assay for pol sequences has been used by several groups and is the most specific PCR assay for XMRV. Three other PCR assays were also used that target the LTR, gag and env regions of XMRV DNA. These assays could detect at least 5 viral copies of XMRV DNA. The precision and reproducibility of the PCR assays, as well as their specificity for XMRV, were also demonstrated. DNA prepared from white blood cells of 100 CFS patients and 200 controls were negative for XMRV. For every 96 PCR reactions, 12 water controls were included; these were always negative for XMRV DNA.
The inability to find XMRV via PCR and antibodies has, of course, happened numerous times before. This time Singh also tried to use culture - for up to six weeks - the time Dr. Mikovits stated was necessary. Unfortunately no viral protein or DNA from XMRV was found either in people in with CFS, healthy controls or people who previously tested positive for XMRV by the WPI.
Infectious XMRV in human plasma. It has been suggested that the most sensitive method for detecting XMRV in patients is to inoculate cultured cells with clinical material and look for evidence of XMRV replication. The XMRV-susceptible cell line LNCaP was therefore infected with 0.1 ml of plasma from 31 patients and 34 healthy volunteers; negative and positive controls were also included. Viral replication was measured by western blot analysis and quantitative PCR. No viral protein or DNA was detected in any culture after incubation for up to 6 weeks.
Then they used the nested PCR techniques used by Lo (which the WPI endorsed). They did find some positives but concluded they came from trace amounts of mouse DNA in their reagents
Presence of mouse DNA. After not finding XMRV using qPCR, serological, and viral culture assays, the authors used the nested PCR assay described by Lo et al. Although positives were observed, they were not consistent between different assays. This led the authors to look for contamination in their PCR reagents. After examination of each component, they found that two different versions of Taq polymerase, the enzyme used in PCR assays, contained trace amounts of mouse DNA.
Dr. Racaniello basically concluded that this study was good enough to conclude that XMRV was not found in these patients and suggested suggested that with regards to researchers the XMRV case was pretty closed but that for patients sake (non-scientists)
the WPI should look to their reagents etc. in order to clear up where the XMRV in their study came from.
Given the care with which these numerous assays were developed and conducted, it is possible to conclude with great certainty that the patient samples examined in this study do not contain XMRV DNA or antibodies to the virus. Its not clear why the 14 patients resampled from the original Lombardi et al. study were negative for XMRV in this new study. The authors suggest one possibility: presence of trace amounts of mouse DNA in the Taq polymerase enzymes used in these previous studies. I believe that it is important to determine the source of XMRV in samples that have been previously tested positive for viral nucleic acid or antibodies. Without this information, questions about the involvement of XMRV in CFS will continue to linger in the minds of many non-scientists.
Dr. Singh, Dr. Bateman and the Lights took a strong stand against the use of antiretrovirals stating that they were 'forced to conclude that prescribing antiretroviral agents to CFS patients is insufficiently justified and potentially dangerous.' and that much data suggests pathogens play a role in CFS and that work should continue.
Given the lack of evidence for XMRV or XMRV-like viruses in our cohort of CFS patients, as well as the lack of these viruses in a set of patients previously tested positive, we feel that that XMRV is not associated with CFS. We are forced to conclude that prescribing antiretroviral agents to CFS patients is insufficiently justified and potentially dangerous.
They also note that there is still a wealth of prior data to encourage further research into the involvement of other infectious agents in CFS, and these efforts must continue.