The patients recruited for the present study were all of Caucasian ethnicity, and were referred from March 2011 to December 2014 to the Cardiology Department of the University Hospital of Pavia (Italy) with suspected CFS. For inclusion in the current study, fatigue must have been present for at least 6 months. Any accompanying symptoms should not have preceded the onset of fatigue and be persistent or recurring for at least 6 months. The current study was in accordance with the guidelines of the Helsinki Declaration for Human Research, and was approved by the ethics committee of the University of Pavia. Subjects exhibiting a chronic disease (such as an allergy, skin disease, vascular disease or diabetes), diagnosis of fibromyalgia, irritable bowel syndrome or using drugs on a regular basis were excluded. Written informed consent was obtained from all the participants.

The entire population underwent the following examinations: Medical history and family anamnesis, blood test, physical examination, blood pressure measurement, echocardiographic examination, basal 12-lead electrocardiogram and continuous electrocardiographic monitoring for 24 h, stress testing cycle ergometer and blood pressure monitoring 24 h. All patients then underwent neurological examination, which established the diagnosis of CFS.

Sociodemographic data included patient age, sex, body mass index and ethnicity. Outcomes of interest regarding onset of the illness included age of onset (age at which the patient first experienced symptoms) and the duration of illness (time in years elapsed since the onset of illness). In addition, the current study accounted for the events and exposure to infectious agents that the participants were subjected to prior to illness.

Exercise stress testing was performed as previously described (8). Briefly, a multistage-bicycle-ergometer exercise stress test was performed with the patients in the supine position with an initial workload of 25 W and successive increments of 25 W every 3 min at a pedalling frequency of 60 rpm. A standard 12-lead electrocardiography was taken before and at the end of each stage, and at the end of the exercise. Leads V4, V5, V6 were monitored during exercise. The systolic and diastolic blood pressures were measured using a sphygmomanometer at basal conditions, at the end of each interval, at the maximum quantity of work and every 3 min in the recovery phase. During the test, the patients were interrogated continuously regarding their symptoms in terms of pain, dyspnea, vertigo and tachycardia, as well as other symptoms. The test was interrupted if one of these conditions manifested: Appearance of chest pain and/or dyspnea, exhaustion, ST segment elevation ≥1 mm without diagnostic Q waves, ST-segment depression ≥2 mm, repetitive arrhythmias or a drop in systolic blood pressure >10 mmHg. The test was considered positive in the presence of down sloping ST-segment depression ≥1 mV below the baseline, at least 0.06–0.08 sec after the QRS complex J point in multiple leads or ST-segment elevation.

Prior to the exercise test, a pharmacological washout was performed. Calcium-channel antagonists and nitrates were halted 48 h before the test. β-Blockers were gradually reduced and subsequently stopped 1 week before the examination. Patients who were taking Digitalis were not included in the current study.

Twenty-four-hour Holter electrocardiogram (ECG) monitoring was performed in all patients enrolled in the study, using a three-channel tape recorder (Cardioline Click Holter Recorder; Cardioline SpA, Trento, Italy). Each tape was manually corrected for noise and ectopic beats. Mean RR intervals and the following HRV parameters were calculated as 24-h values: Standard deviation of all NN interval, square root of the mean of the sum of the square of the differences between adjacent NN intervals and number of adjacent NN intervals differing by >50 msec divided by the total number of all NN intervals (pNN50).

Conventional echocardiography and tissue EcocolorDoppler imaging were performed. M-mode, two-dimensional and Doppler echocardiographic examinations were performed using an ultrasonographic system (MyLab™30Gold Cardiovascular; Esaote SpA, Genoa, Italy) equipped with a multi-frequency transduction. The left ventricle (LV) ejection fraction was calculated from apical two- and four-chamber views using LV volumes according to the modified biplane Simpson's rule. LV end-diastolic volume and LV end-systolic volume were indexed to body surface area. Peak early (E) and late (A) filling velocities, the E:A ratio, and E velocity deceleration time were measured from the LV-inflow pattern at the tips of the mitral valve at the end of expiration. Measurement of systolic pulmonary artery pressure was performed using the maximal regurgitant velocity at the tricuspid valve via continuous wave Doppler.

Continuous variables were expressed as means ± standard deviation for normally distributed data and they were expressed as medians ± standard deviation otherwise. Discrete data were instead analyzed using the χ2 test. P<0.05 was considered to indicate a statistically significant difference.

A total of 44 patients with suspected CFS were included in the current study (9 males and 35 females; mean age, 40±13 years) as presented in Table I.

The mean age of onset of symptoms was 31±13 years. In the current population, the onset of symptoms was fatigue/weakness in nine patients (20%), fever in five patients (11%), flu-like syndrome in two patients (5%), arthromyalgia in one patient (2%), neurological problems in 30 patients (68%), dyspnea in three patients (7%) and viral infectious episodes in seven patients (16%). In addition, one patient developed symptoms following an intestinal infectious episode and one patient developed symptoms following a surgical intervention. The following were considered as neurological problems: Amnesias, cognitive impairment, difficulty concentrating, confusion, headaches and vertigo.

Persistent symptoms included fatigue in 26 patients (59%); unrefreshing sleep in 10 patients (23%); pain (such as headaches) in eight patients (18%); arthromyalgia/myalgia in 14 patients (32%); fever in nine patients (20%), major depression in 11 patients (25%), and mood disorders and anxiety in 12 patients (27%). Two patients (4%) presented with a sore throat and five patients (11%) exhibited lymphadenopathy. The accompanying symptoms observed in these patients underline the heterogeneity of CFS.

Viral serology testing was performed on 3-ml blood samples of all the patients at the time of recruitment and provided evidence of exposure to the Epstein-Barr virus (EBV), cytomegalovirus (CMV) and other human herpesviridae (HHV). In the present study, the presence of past exposure to EBV was observed in 10 patients, seven patients exhibited previous CMV infection, and five patients exhibited antibodies for EBV and CMV, indicative of previous infection with both.

Table II presents the biochemical parameters that were evaluated in the current study. No differences in the concentration of hemoglobin and hematocrit were observed. Furthermore, thyroid function appears to be similar in the two populations, as were the lipid, glucose and creatinine levels. Certain differences were observed in the muscle profiles, but the differences were not statistically significant.

In 19 of the 44 patients with suspected CFS the diagnosis was confirmed according to recent clinical guidelines (9). Of these, three were male and 16 were female (mean age, 40±14 years). The mean age of onset of symptoms was 28±13 years and the cause of this occurrence was viral infection (n=6) and a flu-like episode (n=5). The two groups were comparable regarding sex and gender.

The mean heart rate at baseline in the patients was 79±13 bpm (82±10 bpm in patients with CFS and 77±15 bpm in patients without CFS).

The Holter ECG over 24 h identified the presence of rare/sporadic ventricular or supraventricular arrhythmias in 14 patients. The HRV in patients with and without confirmed diagnosis of CFS were evaluated using ECG monitoring over 24 h. No significant differences were observed for the HRV parameters between the two subpopulations (Table III).

EcocolorDoppler in the study population excluded alteration in left ventricular segmental kinetics, four patients exhibited mitral valve prolapse and the mean ejection fraction was 58±12%. With regard to the patients with confirmed diagnosis of CFS, the mean ejection fraction was 59±4%; in three of these patients with confirmed diagnosis, mitral valve prolapse was observed.

The 24-h blood pressure monitoring demonstrated that nine out of 19 patients with CFS had blood pressure at the lower of the normal limits, and only one with high blood pressure. No symptomatic hypotensive episodes were recorded during the 24-h monitoring period and the nocturnal blood pressure was similar in the two subgroups.