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Metagenomic Investigation of Plasma in Individuals with ME/CFS Highlights the Importance of Technica

hixxy

Senior Member
Messages
1,229
Location
Australia
PLoS One. 2016 Nov 2;11(11):e0165691. doi: 10.1371/journal.pone.0165691. eCollection 2016.

Metagenomic Investigation of Plasma in Individuals with ME/CFS Highlights the Importance of Technical Controls to Elucidate Contamination and Batch Effects.

Miller RR 1, Uyaguari-Diaz M 2, McCabe MN 2, Montoya V 2, Gardy JL 1,2, Parker S 3, Steiner T 4, Hsiao W 5,6, Nesbitt MJ 7, Tang P 8, Patrick DM 1,2; CCD Study Group.

Abstract

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a debilitating disease causing indefinite fatigue. ME/CFS has long been hypothesised to have an infectious cause; however, no specific infectious agent has been identified. We used metagenomics to analyse the RNA from plasma samples from 25 individuals with ME/CFS and compare their microbial content to technical controls as well as three control groups: individuals with alternatively diagnosed chronic Lyme syndrome (N = 13), systemic lupus erythematosus (N = 11), and healthy controls (N = 25). We found that the majority of sequencing reads were removed during host subtraction, thus there was very low microbial RNA content in the plasma. The effects of sample batching and contamination during sample processing proved to outweigh the effects of study group on microbial RNA content, as the few differences in bacterial or viral RNA abundance we did observe between study groups were most likely caused by contamination and batch effects. Our results highlight the importance of including negative controls in all metagenomic analyses, since there was considerable overlap between bacterial content identified in study samples and control samples. For example, Proteobacteria, Firmicutes, Actinobacteria, and Bacteriodes were found in both study samples and plasma-free negative controls. Many of the taxonomic groups we saw in our plasma-free negative control samples have previously been associated with diseases, including ME/CFS, demonstrating how incorrect conclusions may arise if controls are not used and batch effects not accounted for.

PMID: 27806082
DOI: 10.1371/journal.pone.0165691

Pubmed: https://www.ncbi.nlm.nih.gov/pubmed/27806082
Fulltext: http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0165691
 

msf

Senior Member
Messages
3,650
Is this helpful advice for other researchers, or is it basically a mea culpa?
 

RogerBlack

Senior Member
Messages
902
This is basically a negative finding - any signal they found is easily explainable as contamination.
As a general point, it seems not very likely that there is a significant viremea - quantity of circulating virus - long after the initial infection.

It's a useful caution to other researchers.
I note the inadequacy of contamination controls and standards lead to millions of pounds/dollars/... being wasted on the whole XMRV thing.
(which turned out to be a pervasive contamination of lab samples by a virus contaminating cell cultures in laboroatories)
The contamination levels required are ridiculous.
Detecting a drop of blood in a swimming pool is trivial with some of these tests - meaning that contamination is enormously problematic.
 

msf

Senior Member
Messages
3,650
I know it´s a negative finding, I was curious as to whether other groups already take these precautions/allow for these effects or not. I would hope people like Montoya and Lipkin would be aware of these difficulties.
 

barbc56

Senior Member
Messages
3,657
It's unusual to publish negative studies, so this is a breath of fresh air. Researchers are hesitant to publish these studies as they are often perceived as failures. However, negative studies can actually improve what is known. These studies also point out flaws in study design.

More researchers are advocating the importance of publishing negative studies. The article below goes into more detail.

https://www.elsevier.com/connect/scientists-we-want-your-negative-results-too
 

Kati

Patient in training
Messages
5,497
If this is a negative study, then we need to define the parameter. It was negative for bacterial content in the plasma.
They did not test the tissus, they obviously did not test the brains and seemingly did not test for intracellular infections (though I am not entirely sure about the latter, and whether this is technically feasible)

I am not too attached to a particular cause of ME but we have physicians who are convinced we have a persisting enteroviral infection, others who regularily test the cerebro-spinal fluid and find herpes viruses in there.

So I think it would be safer to say that following their particuliar techniques, using plasma, there was no statistically detectable differences.
 

Cheesus

Senior Member
Messages
1,292
Location
UK
So I think it would be safer to say that following their particuliar techniques, using plasma, there was no statistically detectable differences.

This may also be an issue of testing plasma specifically rather than a whole blood sample. In the following study, which also used metagenomic sequencing to investigate the blood microbiome, plasma contained only 0.03% of the total microbial DNA found in the blood of healthy individuals (relevant bit in bold):

Comprehensive description of blood microbiome from healthy donors assessed by 16S targeted metagenomic sequencing.
BACKGROUND:
Recent studies have revealed that the blood of healthy humans is not as sterile as previously supposed. The objective of this study was to provide a comprehensive description of the microbiome present in different fractions of the blood of healthy individuals.

STUDY DESIGN AND METHODS:
The study was conducted in 30 healthy blood donors to the French national blood collection center (Établissement Français du Sang). We have set up a 16S rDNA quantitative polymerase chain reaction assay as well as a 16S targeted metagenomics sequencing pipeline specifically designed to analyze the blood microbiome, which we have used on whole blood as well as on different blood fractions (buffy coat [BC], red blood cells [RBCs], and plasma).

RESULTS:
Most of the blood bacterial DNA is located in the BC (93.74%), and RBCs contain more bacterial DNA (6.23%) than the plasma (0.03%). The distribution of 16S DNA is different for each fraction and spreads over a relatively broad range among donors. At the phylum level, blood fractions contain bacterial DNA mostly from the Proteobacteria phylum (more than 80%) but also from Actinobacteria, Firmicutes, and Bacteroidetes. At deeper taxonomic levels, there are striking differences between the bacterial profiles of the different blood fractions.

CONCLUSION:
We demonstrate that a diversified microbiome exists in healthy blood. This microbiome has most likely an important physiologic role and could be implicated in certain transfusion-transmitted bacterial infections. In this regard, the amount of 16S bacterial DNA or the microbiome profile could be monitored to improve the safety of the blood supply.

https://www.ncbi.nlm.nih.gov/pubmed/26865079

Moreover, the authors of the paper found "At a deeper taxonomic level (Fig. 2C), there are striking differences between fractions," (page 1143) which is to say that there was a significant degree of difference between the microbial populations found in the plasma, buffy coat and red blood cells.

The authors of the study highlighted by @hixxy chose to use plasma for the following reason:

We chose to investigate plasma as this sample type was readily obtainable from both control and case patients and we hypothesized that there would be fewer confounding microbes in plasma compared to more complex sample types such as faeces, making etiologic associations easier to identify.

So, it seems to me that the study was a useful demonstration of the capacity for contamination in shotgun metagenomics, however it was certainly not a useful investigation of blood-borne pathogens in patients with ME.

Having said that, I am not certain of the significance of one study being done on DNA and the other RNA.
 
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HowToEscape?

Senior Member
Messages
626
It's a useful caution to other researchers.
I note the inadequacy of contamination controls and standards lead to millions of pounds/dollars/... being wasted on the whole XMRV thing.
(which turned out to be a pervasive contamination of lab samples by a virus contaminating cell cultures in laboroatories)
The contamination levels required are ridiculous.
Detecting a drop of blood in a swimming pool ......

But didn't the XMRV finding give a basic insight about the current state of lab work? It had no direct use for us but was useful for science. (?)
 

RogerBlack

Senior Member
Messages
902
But didn't the XMRV finding give a basic insight about the current state of lab work? It had no direct use for us but was useful for science. (?)

Yes.
It was pretty much a disaster waiting to happen - the vast cheapening of sequencing meant that things could be done that had never been done before, and the rational testing of all cultures and reagents for contamination hadn't occurred to anyone as a major problem.

It however did seem to get some people interested in CFS, even if their actual results were null because of XMRV not being real.