Celiac Disease Diagnosis in Patients With Weakly Positive Serum Anti-Transglutaminase: Duodenal Anti-Endomysium Assay.
Celiac Disease Diagnosis in Patients With Weakly Positive Serum Anti-Transglutaminase: Duodenal Anti-Endomysium Assay.
Celiac disease (CD) is a chronic immune-mediated disorder that occurs in genetically
 predisposed populations. Patients affected by the disease may be asymptomatic or manifest
 classic malabsorption symptoms of diarrhea, steatorrhea, abdominal pain, and weight loss
 after gluten ingestion (and related derivatives found in other grains). Diagnosis and
 screening begin with the use of serologic tests, i.e. IgA anti-tissue transglutaminase (tTG)
 and IgA anti-endomysial antibodies (EmA). Duodenal biopsy, still considered by many as the
 criterion necessary for diagnosis, demonstrates the pathologic findings of small intestinal
 villous atrophy, crypt hyperplasia, and intraepithelial lymphocytosis that occur on exposure
 to dietary gluten. Genetic tests, revealing permissive haplotypes, may be helpful in
 identifying susceptible individuals. CD diagnosis is still anchored to the criteria
 established by the European Society of Pediatric Gastroenterology Hepatology and Nutrition in
 1990. These require the mandatory presence of (a) villous atrophy with crypt hyperplasia and
 increased intraepithelial lymphocytes (IELs) count when the patient is eating gluten, and (b)
 a full clinical remission after elimination of gluten from the diet. As a consequence,
 patients with minimal or no intestinal histology lesions pose a considerable problem, as
 serum anti-tTG and EmA are known to be often negative, or weakly positive, in patients with
 CD with mild intestinal damage. The investigators, in 2002, measured anti-tTG antibody in the
 culture medium of intestinal biopsy specimens from patients with suspected CD and evaluated
 the relationship between antibody production and severity of intestinal mucosal damage, and
 demonstrated that anti-tTG assay of the culture medium of biopsy specimens can improve the
 accuracy of CD diagnosis in patients negative for serum antibodies. The same investigators,
 in 2011, evaluated the diagnostic accuracy of EmA assay in the culture medium of intestinal
 biopsies for CD diagnosis and demonstrated that EmA assay in the culture medium had a higher
 sensitivity and specificity than serum EmA and anti-tTG assay. The present study is performed
 to investigate the clinical usefulness of the in vitro production of EmA in CD diagnosis in a
 large number of consecutive adult patients with suspected CD and weakly positive [e.g. 2-3xN]
 serum anti-tTG.
Celiac disease (CD) is a chronic immune-mediated disorder that occurs in genetically
 predisposed populations. Patients affected by the disease may be asymptomatic or manifest
 classic malabsorption symptoms of diarrhea, steatorrhea, abdominal pain, and weight loss
 after gluten ingestion (and related derivatives found in other grains). The astute clinician
 must be aware of a more subtle GI picture, as well as non-GI signs and symptoms (e.g.,
 iron-deficiency anemia, abnormal liver function tests, and type 1 diabetes mellitus).
 Diagnosis and screening begin with the use of serologic tests, i.e. immunoglobulin A (IgA)
 anti-tissue transglutaminase (tTG) and IgA anti-endomysial antibodies (EmA). The EmA test is
 performed by indirect immunofluorescence against reagent monkey esophagus or human umbilical
 cord. The EmA bind to the smooth muscle antigen tTG, and the test result is reported as
 titers. EmA are both moderately sensitive and highly specific (sensitivity 85%-98%,
 specificity 97%-100%) for CD, but quickly become negative when the patient starts a
 gluten-free diet. Anti-tTG is a highly sensitive and specific test for CD and is widely
 available as an automated enzyme-linked immunosorbent assay from multiple manufacturers.
 Although the sensitivity and specificity of this test is high, tTG can be present in as many
 as 5% of control patients. In addition, false-positive tTG has been reported in patients with
 Crohn's disease. Duodenal biopsy, still considered by many as the criterion necessary for
 diagnosis, demonstrates the pathologic findings of small intestinal villous atrophy, crypt
 hyperplasia, and intraepithelial lymphocytosis that occur on exposure to dietary gluten.
 These changes exhibit improvement after withdrawal of gluten from the diet. Genetic tests,
 revealing permissive haplotypes, may be helpful in identifying susceptible individuals.

 CD diagnosis is still anchored to the criteria established by the European Society of
 Pediatric Gastroenterology Hepatology and Nutrition in 1990. These require the mandatory
 presence of (a) villous atrophy with crypt hyperplasia and increased intraepithelial
 lymphocytes (IELs) count when the patient is eating gluten, and (b) a full clinical remission
 after elimination of gluten from the diet. However, 20 years after those diagnostic criteria
 were established, there is now growing evidence that at least two other factors must be
 considered. First, symptomatic gluten sensitivity and malabsorption may coexist with a
 normal-looking mucosa. Second, the finding of circulating IgA antibodies to tissue
 transglutaminase (anti-tTG) or endomysium (EmA) at diagnosis is an extremely accurate
 diagnostic instrument. As a consequence, CD diagnosis is nowadays also being done in
 symptomatic patients presenting with a normal-looking mucosa but with a high titer of serum
 EMA and/or anti-tTG antibodies. However, patients with minimal or no intestinal histology
 lesions pose a considerable problem, as serum anti-tTG and EmA are known to be often
 negative, or weakly positive, in patients with CD with mild intestinal damage.

 Picarelli et al., in 1996, tried to establish whether the small intestine of CD patients is
 the site of EmA production and whether gliadin challenge could induce their release. The
 authors evaluated small intestine biopsy samples from treated and untreated CD patients and
 controls, cultured in vitro for 24-48 h in the presence of gliadin. EmA were detected in the
 supernatants of these organ culture biopsy samples by immunofluorescence technique and
 enzyme-linked immunosorbent assay (ELISA), respectively. No EmA were found in the culture
 supernatants of biopsy samples of controls, whereas they were detected in the culture
 supernatants of all untreated CD patients, irrespective of gliadin challenge. Conversely, EmA
 were not detected in supernatants of biopsy samples cultured in medium only from treated CD
 patients, but were detected in the majority of the biopsy samples challenged with gliadin.

 The investigators, in 2002, measured anti-tTG antibody in the culture medium of intestinal
 biopsy specimens from patients with suspected CD and evaluated the relationship between
 antibody production and severity of intestinal mucosal damage, and demonstrated that anti-tTG
 assay of the culture medium of biopsy specimens can improve the accuracy of CD diagnosis in
 patients negative for serum antibodies.

 The same investigators, in 2011, evaluated the diagnostic accuracy of EmA assay in the
 culture medium of intestinal biopsies for CD diagnosis and demonstrated that EmA assay in the
 culture medium had a higher sensitivity and specificity than serum EmA and anti-tTG assay.
 All patients with CD who were tested as false-negatives for serum EmA and/or anti-tTG carried
 the human leukocyte antigen alleles associated to CD. Furthermore, during the follow-up, a
 subgroup of patients with negative-serum EmA/anti-tTG, normal villi architecture, and
 positive-EmA in the culture medium, developed villous atrophy and underwent gluten-free diet,
 with consequent resolution of the symptoms and complete intestinal histology recovery. The
 investigators concluded that EmA assay in the culture medium should be included in the
 diagnostic criteria for CD diagnosis in "seronegative" patients.

 The present study is performed to investigate the clinical usefulness of the in vitro
 production of EmA in CD diagnosis in a large number of consecutive adult patients with
 suspected CD and weakly positive [e.g. normal value multiplied for 2-3 times, 2-3xN] serum
 anti-tTG.
Inclusion Criteria:

 1. adult patients, both genders, aged between 18-70 years;

 2. with suspected CD (i.e. affected with one or more of the following symptoms: chronic
 diarrhea or constipation, alternating bowel habits, abdominal pain, dyspepsia,
 recurrent aphthosis, dental enamel defects, thyroiditis, dermatitis, osteoporosis,
 joints pain, weight loss, anemia, cryptogenetic hypertransaminasemia);

 3. with weakly positive [e.g. 2-3xN] serum anti-tTG antibodies; and d) subjects with a
 family history of CD.

 Exclusion Criteria:

 - patients with IgA deficiency, type 1 diabetes, inflammatory bowel diseases (Crohn's
 disease or ulcerative colitis), Helicobacter pylori infection and other gastrointestinal
 infection, and pregnancy.