Chronic Fatigue Syndrome (CFS) is a complex and poorly understood illness that affects between 1 and 4 million individuals [1, 2] and costs an estimated $35 billion per year in lost productivity and health care [3, 4]. Persistent and unexplained fatigue has been observed as a sequela of acute infection since the first half of the 20th century and the period’s notable outbreaks of infectious disease (e.g., polio epidemics, influenza pandemics). From 1934 to 1984, there were several reports of infectious disease outbreaks simulating poliomyelitis [5, 6] though no causal pathogen was isolated from the majority of the cases. Clinical investigation of individuals who did not recover several years after initial infection showed persistent symptoms of fatigue, sleep disturbance and cognitive impairment [7, 8]. Following an outbreak investigation in 1984 by the Centers for Disease Control and Prevention (CDC) in Incline Village, Nevada , an international group of medical experts defined this protracted post-infection illness as chronic fatigue syndrome (CFS) . Since then, post-infection illness that lasts for more than six months is a central component of CFS, and studies have shown that the incidence of CFS following infection with a number of different pathogens is about 10% [11–14]. The pathogens that most commonly lead to CFS in prospective studies are those that cause infectious mononucleosis (IM)/glandular fever (mainly Epstein-Barr virus (EBV); occasionally cytomegalovirus (CMV), human herpesvirus 6, hepatitis A, and adenovirus , Q fever and Ross River virus (mainly in Australia ), enteroviruses  and parvovirus B19 [16, 17]. Indeed in recent work by Kerr and colleagues, antibody testing for EBV, enterovirus, Coxiella burnetii (causative agent of Q fever) and parvovirus B19 revealed evidence of subtype-specific relationships for EBV and enterovirus, two of the most common infectious triggers of CFS/ME .
There remains therefore an overwhelming body of evidence reinforcing the link to an infectious etiology in at least a subset of CFS patients, despite the fact that specific viral serology are not required for diagnosing CFS [19, 20]. Specifically, there is a notable and long-standing association of CFS with EBV infection. In our previously published analyses of the adolescent cohort presented here [14, 21–24], we reported a diagnosis of CFS at 6, 12 and 24 months following IM, in 13%, 7% and 4%, of the subjects respectively. Ninety percent of CFS subjects at 6 months were female as were all cases at 12 (n = 22) and 24 (n = 13) months following IM. This is consistent with the rates observed by Buchwald and colleagues (2000)  who found 12% of subjects had not recovered and were reporting symptoms of fatigue and impaired functioning 6 months following onset of IM, 76% of whom were female. Nearly identical results were also reported in an Australian cohort where 11% and 9% of subjects satisfied the case definition for CFS at 6 and 12 months following onset of IM, Q fever and Ross River fever (RRF) .
Investigators have sought to understand the underlying causes of these persistent symptoms by surveying immune signaling in the CFS patient population. Early measurements of cytokine concentrations in blood samples from this broader population produced widely discordant results due mainly to differences in case definition and laboratory protocols . Limiting factors also include the focus on a varied and narrow selection of cytokines  as well as a conventional univariate analysis that does not account for the context-specific expression of cytokines . Only one previous study has prospectively evaluated cytokine production in CFS patient populations where a uniform infectious trigger was supported by serology. Hickie et al. (2006)  measured the concentrations of 8 cytokines: IL-1b, IL-2, IL-4, IL-6, IL-10, IL-12, TNF-α, and IFN-γ in serum and culture supernatants of peripheral blood mononuclear cells collected 1, 2, 3, 6 and 12 months following the acute illness (IM, RRF or Q fever). The participants were 22 subjects with confirmed PI-CFS (11 EBV, 6 RRV, and 5 Q fever) and 42 control subjects (17 EBV, 14 RRV, and 11 Q fever) matched for age from the same cohort who had recovered within 6 weeks of symptom onset. The analyses were performed on all PI-CFS subjects as a single group, combining the 11 EBV patients with those infected by a virus uncommon outside Australia. They found no significant differences between PI-CFS cases and recovered controls in serum or culture supernatants . However, serum cytokine levels were almost exclusively below the assay detection limit (8–15 pg/ml) and supernatants were cultured from cryopreserved PBMCs. As a result several unanswered questions remain. For example, do key CFS-specific cytokines remain unmeasured? Perhaps most importantly, could PI-CFS cytokine signatures reflect pathogen-specific (and gender-specific) variations in immune response? If so these may be lost when pooling subjects with different infectious triggers (and different genders).
In an attempt to address some of these questions, this secondary analysis has focused specifically on a group of female adolescents diagnosed PI-CFS as the result of a uniform and known pathogen, namely EBV. An extended survey of 16 cytokines was conducted in plasma using a modified chemiluminescent assay  and identified significant differences in IL-8 and IL-23 concentrations in the patient group at 24 months post-infection. Because cytokines are expressed in a coordinated fashion we also extended the analysis beyond the traditional univariate analysis to investigate combinatorial effects across multiple cytokines. Taken together, a classification of subjects based on levels of IL-2, 6, 8 and 23 supported assignment into the patient or control group with an accuracy exceeding 80% when applied relative to interferon gamma (IFN-γ) concentration. Interestingly the latter did not differ significantly in expression level across subject groups.