Chronic Fatigue Syndrome (CFS), also termed Myalgic Encephalomyelitis, is a severe, systemic illness characterized by debilitating, unexplained, persistent fatigue that indeed has wide impact on the quality of life [1–3]. Besides the disabling fatigue, CFS is accompanied by several symptoms that differ among patients: fatigue not improved by rest; headaches; sleep disturbances; difficulties with concentration; memory problems; muscle weakness; musculoskeletal pain, etc. What is more is that all these symptoms may be aggravated by physical or mental activity [4, 5]. Therefore, CFS is a medically unexplained disorder diagnosed through symptoms reported by the patient and the exclusion of other medical and psychiatric diseases with the same symptoms. The estimated worldwide prevalence of CFS is 0.4-2.5%, with a peak age of onset between 20 and 40 years, and with a female to male ratio of 3:1 .
This clinical disorder is considered a multi-factorial disease with unknown etiology and pathophysiology. Genetic studies have been performed in order to characterize genes that could be useful as CFS biomarkers [7–10]. In particular, it has been proposed that CFS is associated with immunological and inflammatory diseases [7, 8]. The over-expression of pro-inflammatory proteins, such as interferon-γ (INF-γ), interleukin-1 (IL-1), and tumor necrosis factor- α (TNF-α), has been reported in CFS . Besides, up-regulated immune-related genes in patients affected by CFS such as lactotransferrin, defensin-α1, integrins , CMRF35 antigen, IL-8, HD protein , and cathepsin C  are supportive of the notion that CFS is characterized by immune system activation. Nevertheless, at present, there are no specific markers of inflammation and/or immune activation which may be useful in the evaluation of patients with CFS, thus making the treatment more complicated.
Viral infection has been usually proposed as a causative agent, but despite the efforts in the elucidation of the role of virus, results are controversial due to a lack of common standard clinical definition and specific biomarkers of the disease [11–16]. Likewise, vaccines have been depicted by some scholars as playing an important role in CFS onset [17, 18], while on the other hand, others scholars have rejected this possibility, affirming that the vaccine is safe [19, 20].
Whilst in 2011 an International Consensus Panel defined the International Consensus Criteria that identifies the characteristic patterns of the symptom clusters of CFS , the diagnosis is formulated through the patient’s history and exclusion of other medical causes and psychiatric problems . Thereby, the availability of biomarkers for CFS could be of great usefulness for clinical research.
Since genetics is presumed to have a role in the etiology of CFS, one area of investigation is the twin studies which could be useful in elucidating the role of genetic and environmental factors in CFS. Nevertheless, a recent study of Byrnes et al. on monozygotic twins discordant for CFS, did not identify a biomarker for CFS in the transcriptome of peripheral blood leukocytes supposing that positive findings in prior studies may have resulted from experimental bias. Therefore, we moved to the study of proteins, and, for the first time, we used a proteomic approach to evaluate in CFS the global changes of whole saliva (WS).
Recently, the number of publications related to salivary proteome have significantly increased suggesting human saliva as a biological fluid with an enormous potential to reflect systemic health conditions. Saliva has many advantages in terms of low invasiveness, minimum cost, and easy sample collection and processing . Moreover, saliva has a less complex protein composition than serum or plasma reducing the risk of non specific interactions, and at the same time it represents a useful diagnostic tool since about 30% of blood proteins are also present in saliva. Therefore, human saliva proteomics have proven to be a novel approach in the search for protein biomarkers for detection of diseases . In particular, in the last years, we have obtained encouraging results from the proteomic analysis of human WS in rheumatic diseases [24–28]. Hence, with the present study, we investigated the proteomics salivary profile in a couple of monozygotic discordant for CFS. Differences of salivary protein expression in the CFS patient in respect to the healthy co-twin could be strictly correlated to the disease itself if twins differ only regarding the presence of CFS.