Study population and data source

During the 6-year period 2007–2012, we prospectively studied a total of 442 consecutive patients, applying the Rome III criteria for IBS [3] and the ACR 1990 criteria for FMS [6] upon their first visit to the Gastroenterology Outpatient Clinic at the Central University Hospital of Asturias, HUCA (Oviedo, Spain). Most patients were referred from the Rheumatology and Internal Medicine Departments of the same Hospital for the study of a wide variety of long-standing gastrointestinal symptoms.

Diagnosis of IBS was based on a positive history of abdominal discomfort or pain associated with disturbed defecation (Rome III criteria), in the absence of obvious alarm features. Although IBS is no longer a diagnosis of exclusion, since there is good evidence that a positive clinical diagnosis is sufficiently reliable [19], a battery of tests was performed in all patients before commencing the study, a comprehensive medical history was taken, and a thorough physical examination and a complete broad laboratory hematological and biochemical screening were done.

In non-responders to usual therapies, in doubtful cases, or those suspected of having other associated organic illnesses, a specific breath test was performed to exclude possible lactose intolerance or a small bowel bacterial overgrowth. Fecal cultures were performed as needed to rule out parasitic infections in some patients. Furthermore, a total colonoscopy was carried out and random colonic biopsies were taken of patients with persistent diarrhea to rule out microscopic colitis.

An immunological fecal occult blood test (iFOBT) was done in patients aged over 50 years and in those with a positive familial history of colon cancer in first-degree relatives. If this iFOBT was positive, the study was completed with a total colonoscopy.

The inclusion criteria for IBS patients were: (1) Age 18 years or over but less than 65 years; (2) Positive Rome III criteria; (3) Absence of any other associated organic gastrointestinal disease; (4) Negative ACR 1990 criteria for FMS. The same criteria were used to select IBS patients with associated FMS, except the last one, since, by definition, patients had to fulfill the ACR 1990 for FMS classification criteria.

The exclusion criteria for IBS were: (1) Age under 18 or above 65 years; (2) Incomplete or doubtful Rome III criteria; (3) Incomplete or doubtful ACR 1990 criteria for IBS/FMS subjects; (4) Presence of any organic gastrointestinal disease; (5) Any abnormal finding in the analytical screening, colonoscopy or colonic biopsies; (6) Presence of alarm signs or symptoms; (7) Unwillingness to participate in the study.

A total of 263 out of 442 individuals (59%) were eligible to participate in the study. Thirty-four declined to sign the written consent and were excluded. In the end, 229 subjects agreed to take part in this study. Participants were then assigned to one of two groups: the IBS plus FMS group, comprising 104 patients who fulfilled both the Rome III criteria for an IBS diagnosis and the ACR 1990 criteria for FMS classification; the IBS group, comprising the 125 age- and sex-matched, unrelated patients from the same Asturian population who fulfilled the Rome III criteria for IBS diagnosis, but did not exhibit FMS widespread pain or tender points on the skin, and who had negative ACR 1990 criteria.

These 229 subjects were invited to participate on a voluntary basis, after signing a specific informed consent form. The Study Project was approved by the Research and Ethics Committee of the HUCA, following the principles included in the modified Declaration of Helsinki.

Outline of the study protocol

Each volunteer underwent a clinical evaluation, including an updated medical history, a thorough physical examination, a Health-Related Quality of Life (HR-QoL) battery test, and a broad analytical panel.

Duodenal biopsy studies

An upper gastrointestinal endoscopy with at least 4 duodenal biopsies was performed in all the patients included in this case finding/screening, following the usual methodology employed in our Service for CD diagnosis. Samples were routinely stained with Hematoxylin-Eosin (HE) and anti-CD3 immunohistochemical monoclonal antibodies to verify the presence and to account for the number of intraepithelial lymphocytes (IELs). These were in turn quantified per 100 epithelial cells. Sam-ples were studied by two expert pathologists from the HUCA and classified into the following types: Stage 0: Histologically normal duodenum; Stage 1: Increased IEL infiltration with a total count ≥ 25% epithelial cells; Stage 2: Crypt hyperplasia and diffuse chronic inflammatory infiltrate of the lamina propria; Stage 3: Villous atrophy, subdivided into three categories: a) mild, b) moderate, and c) severe, according to the histological classification for CD described by Marsh in 1992 [20] and modified by Oberhüber et al. [21].

Tender points (TPs)

TPs were identified and quantified by digital pressure on the 18 anatomical locations recommended by the ACR 1990 study [6].

Physical, mental, psychological, social functioning and quality of life questionnaires

To measure their physical, mental, psychological, and social functioning, each patient filled out the self-administered Spanish version forms of the Fibromyalgia Impact Questionnaire (FIQ), the Health Assessment Questionnaire (HAQ), and the Short Form Health Survey (SF-36).

FIQ metric

This 10-item instrument measures physical functioning, work status, depression, anxiety, sleep, pain, stiffness, fatigue, and wellbeing on a rating-structured questionnaire, yielding scores between 0 and 80 points. Total FIQ scores are used to define FMS severity, with scores of 0–39, 40–59, and 60–80 corresponding to mild, moderate, and severe FMS, respectively [22].

HAQ metric

The 20-item disability scale of the HAQ measures a patient’s difficulty and need for help and assistive devices in activities of daily living. Each item is scored on a 4-point scale, the highest representing maximum impairment: 0 = able to do without any difficulty, 1 = some difficulty, 2 = much difficulty, and 3 = unable to do [23].

SF-36 metric

The 36 multi-item scale of the short form SF-36 covers 8 aspects of physical and mental health: (1) Physical functioning (PF); (2) Role physical (RF); (3). Bodily pain (BP); (4) General health (GH); (5) Vitality (VT); (6) Social functioning (SF); (7) Role emotional (RE); (8) Mental health (MH).

The scores of these 8 items are aggregated into two weighted, norm-based Physical and Mental Component Summaries (PCS and MCS, respectively), which take values from 0 (poorest health) to 100 (best health status). No reliable relationship has been established between presence and severity of disease and SF-36 scores. For guidance, the published mean values (and standard deviations) for adults in the general Spanish population are 73.0 ± 27.8 (PCS) and 74.4 ± 24.4 (MCS) [24].

To evaluate the severity of digestive symptoms and the amount of pain and fatigue experienced by the patients we used three appropriate types of Visual Analogue Scales (VAS) [25–27].

Cell blood count and coagulation studies were performed with an automated Abbott Hematology Analyzer (Cell Dyn 3500), and the Coagulation Analyzer ACL 3000 (Menarini), respectively. Biochemical tests were performed on a Hitachi Modular automated analyzer SXA-PPBD (Roche) using enzymatic or kinetic methods to measure urea, glucose, total protein, albumin, C-reactive protein, calcium, folate, vitamin B-12, creatinine, creatine kinase, rheumatoid factor, lipid profile, liver function, IgG, IgA and IgM immunoglobulins, iron metabolism, thyroid function, and to carry out a urinalysis with microscopic examination of the sediment.

Anti-nuclear antibodies (ANAs) and anti-thyroid peroxidase (anti-TPO) antibodies were measured in each participant. In cases with altered liver function test results, anti-mitochondrial antibodies (AMAs) were assessed by indirect immunofluorescence assay in the Hep-20-10 cell line (Euroimmun, Lübeck, Germany).

Anti-IgA tissue Trans-Glutaminase subtype 2 (tTG) antibodies were measured with an ELISA kit from Phadia Diagnostics (Upsala, Sweden).

HLA-DQ2 genetic markers (DQA1*0501 and DQB1*0201 alleles) were determined by a polymerase chain reaction (PCR) with a Protrans® HLA Celiac Disease Domino System (Protrans, Ketsch, Germany) kit. The HLA-DQ8 haplotype was characterized in an HLA-DQ2-negative case with villous atrophy.

Statistical analysis

Descriptive statistics were derived for continuous variables (mean, standard deviation, and range) and categorical variables (percentages). Normally distributed continuous outcome measures were analyzed using Student’s t tests, or ANOVA followed by a post hoc Fisher’s test, as appropriate. The chi square contingency test (or Fisher’s exact test where appropriate) was used to analyze categorical data. All statistical tests were carried out using SPSS 15.0 (SPSS Inc, Chicago, IL, 2009). Two-sided P values < 0.05 were considered to be statistically significant.