**3. Timeline**

#### **3.1. April 2000**

An Irish pathologist, John O'Leary, reports "scientific results in a series of children with au‐ tistic enterocolitis… following an approach made by Andrew Wakefield" before the US Congress Committee on Government Reform [5]. He had "compelling" evidence "in relation to the presence of measles virus in children with autistic enterocolitis" and could "confirm that his *[Wakefield's]* hypothesis is correct". This statement was based on the detection of MeV using the then rather novel reverse transcription (RT)-qPCR assay as well other meth‐ ods, in 24/25 "children with autistic enterocolitis", compared to 1/15 control children. O'Leary also emphasised that he went to "desperate lengths" to prove the absence of con‐ tamination problems to "outrule the possible generation of false positives". Importantly, he stressed that "nothing in [his] testimony should or must be construed as anti-vaccine; rather it encourages safe vaccine strategies". This final qualification, although very clear, was bur‐ ied by the headline news of the link between MeV and "autistic enterocolitis".

**3.4. May 2004**

**3.5. Summer 2004**

**3.6. June 2007**

**3.7. February 2009-August 2010**

**4. Analysis of the 2002 O'Leary paper**

An abstract entitled "TaqMan RT-PCR Detection of Measles Virus Genomic RNA in Cere‐ brospinal Fluid in Children with Regressive Autism" and published on the website of the Association of American Physicians and Surgeons lists two of the authors from the 2002 publication as co-authors, with JJ O'Leary occupying the senior author position[8]. It reports the detection of MeV in the cerebrospinal fluid of 19/28 children presenting "as autistic re‐ gression closely following MMR vaccination" compared with 1/37 controls. In five cases a haemaglutinnin*[sic]* gene allelic discrimination assay was carried out and showed a result consistent with a vaccine strain being detected. The abstract concludes with the sentence: "The findings confirm a highly significant statistical association between the presence of MV

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A paper is published that includes six patients (three patients, three controls) whose data were already published in the May 2004 abstract. It reports the detection of MeV in 3/3 pa‐ tients but 0/3 controls [9]. Interestingly, O'Leary and Sheils are not listed as authors; instead

The first of three omnibus autism proceedings took place at the US Court of Federal Claims, where all vaccine claims are managed and are adjudicated by the Office of Special Masters. The trials were designed to establish whether or not autism could be caused by thimerosal containing vaccines, by MMR vaccine, or a combination of the two. I acted as an expert wit‐ ness for the US Department of Justice (DoJ), presenting evidence based on earlier reports I had prepared for the UK High Court, who gave permission for the release to the US Secreta‐ ry of the Department of Health and Human Services of two reports authored by myself. These documents had been filed by the three principal defendants in the UK MMR vaccine litigation and constituted an exhaustive analysis of the raw data underlying the results re‐ ported by the 2002 O'Leary publication. The evidence presented to the trial is a matter of public record, with the transcript available from the autism omnibus proceedings[10].

The evidence presented at the 2007 trial was used in further trials that concluded in February 2009 that there was no credible link between the MMR vaccine and autism. These decisions were upheld on appeal in July/August 2009 and then again in August 2010 were read [11].

The analyses and conclusions shown below were reached after examining all the RT-qPCR data disclosed following a court order. New experimental reports were generated using

RNA in CSF and autistic regression following MMR vaccination".

the publication only thanks them in the acknowledgements.

#### **3.2. August 2000**

A brief letter signed by JJ O'Leary, V Uhlmann and AJ Wakefield appeared in the Lancet [6]. It asserted that their "data from molecular virological studies examining the role of measles vi‐ rus infection in children with autism and enterocolitis have been peer-reviewed, presented, and published at four international scientific meetings". The letter contained references to oth‐ er publications, with reference 4 listing a publication by Uhlmann *et al*. entitled "Identification of measles virus genomes in ileo-colonic lymphoid hyperplasia in children" as "in press" in the Journal Laboratory Investigations. However, there is no record of such a publication.

#### **3.3. April 2002**

Speculation about a possible association between intestinal abnormalities in children with developmental disorders and the MMR vaccine was encouraged by a publication that utilis‐ ed RT-qPCR assays to screen children's intestinal biopsies for the presence of MeV [7]. A comparison of terminal ileal samples from 70 normal controls and 91 children with a "new form of developmental disorder, ileocoloniclymphonodular hyperplasia", led to the claim that whereas 75/91 of the affected children patients tested positive for MeV, only 5/70 con‐ trol patients did. The paper does not reveal whether the autistic children had been given the MMR vaccination, but in the context of the source (Royal Free Hospital), authors (including Wakefield), introduction (reference to Wakefield's paper) and the discussion one is left with the impression that they had. The prominently displayed "take home message" concludes "the data confirmed an association between the presence of measles virus and gut pathology in children with developmental disorder". These results, if true, would constitute hard evi‐ dence linking MeV, gut pathology and autism and was indeed used to support the vaccina‐ tion/autism theory, even though the authors themselves never made that specific link.

#### **3.4. May 2004**

**3. Timeline**

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**3.1. April 2000**

**3.2. August 2000**

**3.3. April 2002**

An Irish pathologist, John O'Leary, reports "scientific results in a series of children with au‐ tistic enterocolitis… following an approach made by Andrew Wakefield" before the US Congress Committee on Government Reform [5]. He had "compelling" evidence "in relation to the presence of measles virus in children with autistic enterocolitis" and could "confirm that his *[Wakefield's]* hypothesis is correct". This statement was based on the detection of MeV using the then rather novel reverse transcription (RT)-qPCR assay as well other meth‐ ods, in 24/25 "children with autistic enterocolitis", compared to 1/15 control children. O'Leary also emphasised that he went to "desperate lengths" to prove the absence of con‐ tamination problems to "outrule the possible generation of false positives". Importantly, he stressed that "nothing in [his] testimony should or must be construed as anti-vaccine; rather it encourages safe vaccine strategies". This final qualification, although very clear, was bur‐

A brief letter signed by JJ O'Leary, V Uhlmann and AJ Wakefield appeared in the Lancet [6]. It asserted that their "data from molecular virological studies examining the role of measles vi‐ rus infection in children with autism and enterocolitis have been peer-reviewed, presented, and published at four international scientific meetings". The letter contained references to oth‐ er publications, with reference 4 listing a publication by Uhlmann *et al*. entitled "Identification of measles virus genomes in ileo-colonic lymphoid hyperplasia in children" as "in press" in the

Speculation about a possible association between intestinal abnormalities in children with developmental disorders and the MMR vaccine was encouraged by a publication that utilis‐ ed RT-qPCR assays to screen children's intestinal biopsies for the presence of MeV [7]. A comparison of terminal ileal samples from 70 normal controls and 91 children with a "new form of developmental disorder, ileocoloniclymphonodular hyperplasia", led to the claim that whereas 75/91 of the affected children patients tested positive for MeV, only 5/70 con‐ trol patients did. The paper does not reveal whether the autistic children had been given the MMR vaccination, but in the context of the source (Royal Free Hospital), authors (including Wakefield), introduction (reference to Wakefield's paper) and the discussion one is left with the impression that they had. The prominently displayed "take home message" concludes "the data confirmed an association between the presence of measles virus and gut pathology in children with developmental disorder". These results, if true, would constitute hard evi‐ dence linking MeV, gut pathology and autism and was indeed used to support the vaccina‐ tion/autism theory, even though the authors themselves never made that specific link.

Journal Laboratory Investigations. However, there is no record of such a publication.

ied by the headline news of the link between MeV and "autistic enterocolitis".

An abstract entitled "TaqMan RT-PCR Detection of Measles Virus Genomic RNA in Cere‐ brospinal Fluid in Children with Regressive Autism" and published on the website of the Association of American Physicians and Surgeons lists two of the authors from the 2002 publication as co-authors, with JJ O'Leary occupying the senior author position[8]. It reports the detection of MeV in the cerebrospinal fluid of 19/28 children presenting "as autistic re‐ gression closely following MMR vaccination" compared with 1/37 controls. In five cases a haemaglutinnin*[sic]* gene allelic discrimination assay was carried out and showed a result consistent with a vaccine strain being detected. The abstract concludes with the sentence: "The findings confirm a highly significant statistical association between the presence of MV RNA in CSF and autistic regression following MMR vaccination".

#### **3.5. Summer 2004**

A paper is published that includes six patients (three patients, three controls) whose data were already published in the May 2004 abstract. It reports the detection of MeV in 3/3 pa‐ tients but 0/3 controls [9]. Interestingly, O'Leary and Sheils are not listed as authors; instead the publication only thanks them in the acknowledgements.

#### **3.6. June 2007**

The first of three omnibus autism proceedings took place at the US Court of Federal Claims, where all vaccine claims are managed and are adjudicated by the Office of Special Masters. The trials were designed to establish whether or not autism could be caused by thimerosal containing vaccines, by MMR vaccine, or a combination of the two. I acted as an expert wit‐ ness for the US Department of Justice (DoJ), presenting evidence based on earlier reports I had prepared for the UK High Court, who gave permission for the release to the US Secreta‐ ry of the Department of Health and Human Services of two reports authored by myself. These documents had been filed by the three principal defendants in the UK MMR vaccine litigation and constituted an exhaustive analysis of the raw data underlying the results re‐ ported by the 2002 O'Leary publication. The evidence presented to the trial is a matter of public record, with the transcript available from the autism omnibus proceedings[10].

#### **3.7. February 2009-August 2010**

The evidence presented at the 2007 trial was used in further trials that concluded in February 2009 that there was no credible link between the MMR vaccine and autism. These decisions were upheld on appeal in July/August 2009 and then again in August 2010 were read [11].

#### **4. Analysis of the 2002 O'Leary paper**

The analyses and conclusions shown below were reached after examining all the RT-qPCR data disclosed following a court order. New experimental reports were generated using identical analysis software, compared with those disclosed by the O'Leary laboratory and any differences were noted. Any ambiguous or discordant results and all results involving negative controls were further investigated by scrutinising the raw data collected by the qPCR instrument. This permitted a definitive resolution of all ambiguities. All disclosed op‐ erator sheets and laboratory notebook entries relevant to the RT-qPCR assay were red and compared with the disclosed and the reanalysed data. Standard operating procedures were examined and inconsistencies with actual procedures were noted.

a FFPE sample. This is in fact the results the authors obtain for the control RNA, where there was an approximately 4,000-fold reduction in RNA levels (Figure 3A). In contrast, the results recorded for MeV RNA were the same regardless of its source (Figure 3B).

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**Figure 3.** FFPE vs fresh samples. A. Effects of formalin fixation on control gene expression levels showing the differ‐ ence caused by the formalin fixation process. Due to the exponential nature of the PCR reaction, the difference be‐ tween the average quantification cycle recorded for fresh tissue and that for formalin-fixed tissue (25 vs 37) equates to 212 or a 4,000-fold reduction of target. B. Absence of an effect of formalin fixation on MeV RNA, with the average

Since any RNA present during formalin fixation would have been affected in an identical manner, the obvious implication of these results is that whilst the control RNA was indeed present prior to formalin fixation and so was degraded, the MeV target was not degraded and entered the sample after formalin fixation. Consequently, no MeV RNA can have been

**b.** Detection of an internal control following RNA extraction is a useful indicator of wheth‐ er the extraction has been successful: absence of amplification implies that it has not been. Consequently, the O'Leary laboratory standard operating procedures implement‐ ed such an assessment and prescribed the exclusion of such samples from further analy‐ sis (Figure 4A). Fortuitously from the investigator's point of view and against the rules

present in the tissue and the positive result must have been caused by a contaminant.

quantification cycles very similar.

**1.** Transparency of reporting

The purpose of publishing a paper in the peer-reviewed literature is to provide ade‐ quate information that allows any competent scientist to follow the published protocol and reproduce the published data. Hence it is essential that detailed descriptions of the methods used and of the results obtained are included. It is not acceptable to publish summarised results only without any supporting, relevant data.


a FFPE sample. This is in fact the results the authors obtain for the control RNA, where there was an approximately 4,000-fold reduction in RNA levels (Figure 3A). In contrast, the results recorded for MeV RNA were the same regardless of its source (Figure 3B).

identical analysis software, compared with those disclosed by the O'Leary laboratory and any differences were noted. Any ambiguous or discordant results and all results involving negative controls were further investigated by scrutinising the raw data collected by the qPCR instrument. This permitted a definitive resolution of all ambiguities. All disclosed op‐ erator sheets and laboratory notebook entries relevant to the RT-qPCR assay were red and compared with the disclosed and the reanalysed data. Standard operating procedures were

The purpose of publishing a paper in the peer-reviewed literature is to provide ade‐ quate information that allows any competent scientist to follow the published protocol and reproduce the published data. Hence it is essential that detailed descriptions of the methods used and of the results obtained are included. It is not acceptable to publish

**a.** RNA was extracted from fresh frozen samples as well as formalin fixed, paraffin-em‐ bedded tissue (FFPE). However, there is no information on how the fresh samples were frozen, how long they had been stored,what percentage of patient and control samples were fresh frozen or FFPE and whether the same percentage was in each category. This is essential, since it is well established that FFPE treatment modifies and destroys RNA, or in Prof O'Leary's own words "wax and fixation by itself breaks down RNA" [5]. Hence it was well known at that time that RNA-derived data obtained from FFPE sam‐

**b.** No information is provided with respect to quantification or quality assessment of the extracted RNA; indeed there is no mention of RNA quality. This is vital information

**c.** The RT-qPCR results are summarised without providing any actual data; a table simply states that 70/91 children with gut pathology were positive for MeV, as against 4/70 in

ies of RNA/ng total RNA; no corresponding figure is provided for the four positive samples from the control samples. There is also no indication of what the potential error

**e.** Despite claims that the authors looked at two viral gene targets, they used only the data

**a.** RNA was extracted from both fresh frozen and FFPE tissue samples and subjected to RT-qPCR analysis. Two RNAs were targeted; a control mRNA specified by the GAPDH gene and the MeV F-gene. As discussed earlier, since FFPE samples are characterised by RNA degradation, the expectation is that the results obtained from the FFPE samples should be different and, in terms of quantification, there should be less RNA present in

cop‐

**d.** MeV copy numbers in the affected children are reported as ranging from 1 to 3x105

needed to assess the validity of any quantitative or negative result [15].

from the F-gene, which were discordant with the H-gene results.

examined and inconsistencies with actual procedures were noted.

summarised results only without any supporting, relevant data.

ples must be analysed and interpreted with caution [12-14].

**1.** Transparency of reporting

88 Recent Advances in Autism Spectrum Disorders - Volume I

the control group.

in those copy numbers might be.

**2.** Reliability of protocols and techniques

**Figure 3.** FFPE vs fresh samples. A. Effects of formalin fixation on control gene expression levels showing the differ‐ ence caused by the formalin fixation process. Due to the exponential nature of the PCR reaction, the difference be‐ tween the average quantification cycle recorded for fresh tissue and that for formalin-fixed tissue (25 vs 37) equates to 212 or a 4,000-fold reduction of target. B. Absence of an effect of formalin fixation on MeV RNA, with the average quantification cycles very similar.

Since any RNA present during formalin fixation would have been affected in an identical manner, the obvious implication of these results is that whilst the control RNA was indeed present prior to formalin fixation and so was degraded, the MeV target was not degraded and entered the sample after formalin fixation. Consequently, no MeV RNA can have been present in the tissue and the positive result must have been caused by a contaminant.

**b.** Detection of an internal control following RNA extraction is a useful indicator of wheth‐ er the extraction has been successful: absence of amplification implies that it has not been. Consequently, the O'Leary laboratory standard operating procedures implement‐ ed such an assessment and prescribed the exclusion of such samples from further analy‐ sis (Figure 4A). Fortuitously from the investigator's point of view and against the rules of their own SOP, the authors did not discard all samples where the control had been negative. Instead, they reported positive MeV results from samples that contained RNA as well as in autistic patient samples that had been negative for their control (Figure 4B). Since, these samples do not contain RNA by their own definition, their test must be detecting a contaminant.

**c.** Two tests accidentally omitted including the RT step before the PCR test. In the case of the control, the results are as expected: the assay works significantly less well (Figure 5A). This is because *Taq* polymerase is very inefficient at making DNA from RNA. In contrast, the four MeV samples tested give the same result, regardless, indicating that the test is detecting DNA (Figure 5B). Since MeV does not exist as DNA, the test is not

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**Figure 5.** Absence of RT step. A. Control RNA: in the absence of the RT step (no RT), no amplification is observed (a Cq of 40/45 equates by definition to no amplification). B. Measles "RNA": in the absence of the RT step, amplification is

**d.** Successful qPCR amplification is characterised by a characteristic "amplification plot", as obtained from sample F4 in Figure 6A and highlighted by the solid arrow. However a second sample (H2) is also recorded as generating a positive result (circled) even though it is clearly not being amplified (dashed arrow). The correct result is obtained by moving the threshold line(dotted arrow) so that well F4 continues to record a positive results, whereas well H2 now records a Cq of 40, which in this setup equates to "no am‐

observed with Cqs in the same range as in the presence of the RT step (RT).

plification" (Figure 6B). This is a rather elementary mistake.

detecting MeV but a DNA contaminant.

**Figure 4.** Workflow according to the O'Leary SOP. A. Following RNA extraction, only samples testing positive for GAPDH should have been further analysed for two viral targets. Samples testing negative should have been discarded and fresh RNA extractions attempted. B. MeV target detection from control+ve samples and control-ve samples show‐ ing that there is no difference in the quantification cycles.

**c.** Two tests accidentally omitted including the RT step before the PCR test. In the case of the control, the results are as expected: the assay works significantly less well (Figure 5A). This is because *Taq* polymerase is very inefficient at making DNA from RNA. In contrast, the four MeV samples tested give the same result, regardless, indicating that the test is detecting DNA (Figure 5B). Since MeV does not exist as DNA, the test is not detecting MeV but a DNA contaminant.

of their own SOP, the authors did not discard all samples where the control had been negative. Instead, they reported positive MeV results from samples that contained RNA as well as in autistic patient samples that had been negative for their control (Figure 4B). Since, these samples do not contain RNA by their own definition, their test must be

**Figure 4.** Workflow according to the O'Leary SOP. A. Following RNA extraction, only samples testing positive for GAPDH should have been further analysed for two viral targets. Samples testing negative should have been discarded and fresh RNA extractions attempted. B. MeV target detection from control+ve samples and control-ve samples show‐

ing that there is no difference in the quantification cycles.

detecting a contaminant.

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**Figure 5.** Absence of RT step. A. Control RNA: in the absence of the RT step (no RT), no amplification is observed (a Cq of 40/45 equates by definition to no amplification). B. Measles "RNA": in the absence of the RT step, amplification is observed with Cqs in the same range as in the presence of the RT step (RT).

**d.** Successful qPCR amplification is characterised by a characteristic "amplification plot", as obtained from sample F4 in Figure 6A and highlighted by the solid arrow. However a second sample (H2) is also recorded as generating a positive result (circled) even though it is clearly not being amplified (dashed arrow). The correct result is obtained by moving the threshold line(dotted arrow) so that well F4 continues to record a positive results, whereas well H2 now records a Cq of 40, which in this setup equates to "no am‐ plification" (Figure 6B). This is a rather elementary mistake.

**3.** Interpretation of control results

results from this run.

**Figure 7.** Contamination (1). A. The report submitted by the O'Leary laboratory shows a single negative NTC (circled), with well E4 next to the NTC not analysed, despite all other samples having been analysed in duplicate. B. A re-analysis that includes well E4 shows that this well is contaminated (arrow) and generates a reading (circled), invalidating any

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**Figure 6.** Inappropriate analysis. A. The data as reported by the O'Leary laboratory. B. The re-analysed data showing absence of amplification in well H2 (circled).

**3.** Interpretation of control results

**Figure 6.** Inappropriate analysis. A. The data as reported by the O'Leary laboratory. B. The re-analysed data showing

absence of amplification in well H2 (circled).

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**Figure 7.** Contamination (1). A. The report submitted by the O'Leary laboratory shows a single negative NTC (circled), with well E4 next to the NTC not analysed, despite all other samples having been analysed in duplicate. B. A re-analysis that includes well E4 shows that this well is contaminated (arrow) and generates a reading (circled), invalidating any results from this run.

**b.** Figure 8A shows a similar situation, with all analyses except the NTC in well B11 car‐ ried out in duplicate. Again, these were the data disclosed to the investigation, and I had to await the release of the raw data runs to be able to re-analyse those results. This time, a re-analysis of the data including well B12 shows both tests gave positive results, ie both negative controls reported contamination of the test (Figure 8B). The only con‐ clusion from these data can be that the data are unreliable.

**4.** Reproducibility of the data

**5. The MIQE guidelines**

manuscript to enhance critical appraisal.

their own, they have never retracted their original paper.

There have been a number of studies attempting to reproduce the findings of the 2002 paper [16-18]. All failed to do so; instead they provided strong evidence for contamination being the cause of the positive findings. However, there were some technical differences between the studies in the choice of tissue (intestine vs blood) or protocols (enzymes, qPCR chemis‐ tries). Therefore, whilst there was a strong suggestion that Prof O'Leary's laboratory was de‐ tecting contaminants, there was no proof. However, any lingering doubt evaporated with the publication from a multi-centre group of authors that refuted any association between persistent MeV RNA in the gut and autism[19]. Astonishingly, this publication includes the two main authors of the Uhlmann paper, and despite publishing evidence that contradicts

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The problems inherent in the design and reporting of qPCR-based assays had been known for a long time, but there had been no concerted effort to tackle this serious problem. The 2002 O'Leary paper is not unique in omitting sufficient experimental detail, which impedes the readers' ability to evaluate critically the quality of the results presented, repeat the re‐ ported experiments, or integrate methodological advances into their own studies. Indeed, a recent survey of qPCR-based publication demonstrates very clearly that this problem per‐ sists even in 2010 [20] and that the perceived quality of the publishing journal is not correlat‐ ed with the actual quality of the qPCR publication. The egregious use of qPCR in the MeV/MMR/autism context provided the stimulus to initiate a push towards improving the technical aspects of qPCR assay design and reporting. Consequently, guidelines tackling this issue have recently been published which aim to promote consistency between laboratories, and increase experimental transparency [21]. The Minimum Information for publication of Quantitative real-time PCR Experiments (MIQE) guidelines outline the minimum data set necessary to evaluate effectively RT-qPCR assays, and are designed to be used as a checklist to both accompany manuscript submission and to be available alongside the published

The four key areas of standardisation that define any qPCR experiment are study design, technical detail, analysis methods and statistics. MIQE addresses these under a set of cap‐ tions that describe a large number of individual elements: "Experimental design, sample, nucleic acids, reverse transcription, target, primers and probes, assay details, PCR cycling and data analysis". At first sight, these look daunting, arduous and over-exacting. In prac‐ tice, it is clear that most, if not all of these parameters describe information that would be obtained as a matter of course during the experimental design, optimisation and validation stages. Importantly, there is a clear hierarchy with some parameters, labelled "E" (essential) in the published guidelines, indispensable for an adequate description of the qPCR assay, whereas other components, labelled "D" (desirable) more peripheral, yet constituting an ef‐ fective foundation for the realisation of best practice protocols. There is increasing recogni‐

**Figure 8.** Contamination (2). A. The report submitted by the O'Leary laboratory shows a single negative NTC at B11, with well B12 next to it not analysed, despite all other samples having been analysed in duplicate. B. A re-analysis that includes well B12 shows that both NTCs are contaminated (circled), invalidating any results from this run.

#### **4.** Reproducibility of the data

**b.** Figure 8A shows a similar situation, with all analyses except the NTC in well B11 car‐ ried out in duplicate. Again, these were the data disclosed to the investigation, and I had to await the release of the raw data runs to be able to re-analyse those results. This time, a re-analysis of the data including well B12 shows both tests gave positive results, ie both negative controls reported contamination of the test (Figure 8B). The only con‐

**Figure 8.** Contamination (2). A. The report submitted by the O'Leary laboratory shows a single negative NTC at B11, with well B12 next to it not analysed, despite all other samples having been analysed in duplicate. B. A re-analysis that

includes well B12 shows that both NTCs are contaminated (circled), invalidating any results from this run.

clusion from these data can be that the data are unreliable.

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There have been a number of studies attempting to reproduce the findings of the 2002 paper [16-18]. All failed to do so; instead they provided strong evidence for contamination being the cause of the positive findings. However, there were some technical differences between the studies in the choice of tissue (intestine vs blood) or protocols (enzymes, qPCR chemis‐ tries). Therefore, whilst there was a strong suggestion that Prof O'Leary's laboratory was de‐ tecting contaminants, there was no proof. However, any lingering doubt evaporated with the publication from a multi-centre group of authors that refuted any association between persistent MeV RNA in the gut and autism[19]. Astonishingly, this publication includes the two main authors of the Uhlmann paper, and despite publishing evidence that contradicts their own, they have never retracted their original paper.
