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Analysis of 16S rDNA sequences and circulating cell-free DNA concentration from plasma of a chronic fatigue syndrome and non fatigued subjects.

Vernon SD, Shukla S, Unger ER, Reeves WC.
Analysis of 16S rDNA sequences and circulating cell-free DNA concentration from plasma of a chronic fatigue syndrome and non fatigued subjects.
Biomed Central Microbiology 2:39, 2002.

Summary

Although many studies have failed to show a consistent association between specific infectious agents and CFS, there remains substantial evidence implicating that microbial organisms play a role in the pathogenesis of CFS. New molecular techniques, such as the 16S rDNA polymerase chain reaction (PCR) assay have not been applied to studies of CFS. This assay relies on the amplification of the gene coding for ribosomal RNA (16S rRNA), which is present in all bacteria and rickettsia, but is absent in eukaryotes, including humans (e.g., blood cells). Amplification is followed by identification of the products. This assay has several advantages over traditional microbiological methods. It can detect infections by uncultivable pathogens where routine microbiological techniques have failed to detect the presence of bacteria in the clinical samples. In addition, when combined with DNA sequencing the assay provides a definite identification of the infectious agents. The 16S rRNA contains conserved and highly divergent regions. Conserved regions permit the design of broad range PCR primers that will find its target in most bacteria. Specific primers can then be used to amplify intervening unique signature sequences, which provide the basis of bacterial identification. This method is increasingly being used in many clinical microbiological laboratories to detect and identify known, novel, nonviable, and uncultivable pathogens from clinical samples. This study tested for 16S rRNA sequences in 34 CFS patients and 55 non-fatigued controls. We failed to find evidence that specific 16S rRNA sequences were associated with CFS.

Abstract

Background: The association of an infectious agent with chronic fatigue syndrome (CFS) has been difficult and is further complicated by the lack of a known lesion of diseased tissue. Cell-free plasma DNA could serve as a sentinel of infection and disease occurring throughout the body. This type of systemic sample coupled with broad-range amplification of bacterial sequences was used to determine whether a bacterial pathogen was associated with CFS. Plasma DNA from 34 CFS and 55 non-fatigued subjects was assessed to determine plasma DNA concentration and the presence of bacterial 16S ribosomal DNA (rDNA) sequences.

Results: DNA was isolated from 81 (91%) of 89 plasma samples. The 55 non-fatigued subjects had higher plasma DNA concentrations than those with CFS (average 151 versus 91 ng) and more CFS subjects (6/34, 18%) had no detectable plasma DNA than non-fatigued subjects (2/55, 4%), but these differences were not significant. Bacterial sequences were detected in 23 (26%) of 89. Only 4 (14%) CFS subjects had 16S rDNA sequences amplified from plasma compared with 17 (32%) of the non-fatigued (P = 0.03). All but 1 of the 23 16S rDNA amplicon-positive subjects had five or more unique sequences present.

Conclusions: CFS subjects had slightly lower concentrations or no detectable plasma DNA than non-fatigued subjects. There was a diverse array of 16S rDNA sequences in plasma DNA from both CFS and non-fatigued subjects. There were no unique, previously uncharacterized or predominant 16S rDNA sequences in either CFS of non-fatigued subjects.

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