- Email: [email protected]
Diagnosis and classification of celiac disease and gluten sensitivity
Autoimmunity Reviews 13 (2014) 472–476
Contents lists available at ScienceDirect
Autoimmunity Reviews journal homepage: www.elsevier.com/locate/autrev
Review
Diagnosis and classification of celiac disease and gluten sensitivity Elio Tonutti a, Nicola Bizzaro b,⁎ a b
Immunopathology and Allergology, Azienda Ospedaliero-Universitaria, Piazza S. Maria della Misericordia 15, 33100 Udine, Italy Laboratory of Clinical Pathology, San Antonio Hospital, via Morgagni 18, 33028 Tolmezzo, UD, Italy
a r t i c l e
i n f o
a b s t r a c t
Article history: Accepted 13 November 2013 Available online 15 January 2014
Celiac disease is a complex disorder, the development of which is controlled by a combination of genetic (HLA alleles) and environmental (gluten ingestion) factors. New diagnostic guidelines developed by ESPGHAN emphasize the crucial role of serological tests in the diagnostic process of symptomatic subjects, and of the detection of HLA DQ2/DQ8 alleles in defining a diagnosis in asymptomatic subjects belonging to at-risk groups. The serological diagnosis of CD is based on the detection of class IgA anti-tissue transglutaminase (anti-tTG) and antiendomysial antibodies. In patients with IgA deficiency, anti-tTG or anti-deamidated gliadin peptide antibody assays of the IgG class are used. When anti-tTG antibody levels are very high, antibody specificity is absolute and CD can be diagnosed without performing a duodenum biopsy. Non-celiac gluten sensitivity is a gluten reaction in which both allergic and autoimmune mechanisms have been ruled out. Diagnostic criteria include the presence of symptoms similar to those of celiac or allergic patients; negative allergological tests and absence of anti-tTG and EMA antibodies; normal duodenal histology; evidence of disappearance of the symptoms with a gluten-free diet; relapse of the symptoms when gluten is reintroduced. © 2014 Elsevier B.V. All rights reserved.
Keywords: Celiac disease Diagnostic criteria Gluten sensitivity Anti-tissue transglutaminase antibodies HLA DQ2/DQ8
Contents 1. Introduction . . . . . 2. Pathogenesis . . . . . 3. Epidemiology . . . . 4. Clinical manifestations 5. Antibodies . . . . . . 6. Intestinal biopsy . . . 7. Diagnostic criteria . . References . . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
1. Introduction Celiac disease (CD) is a chronic, immune-mediated, gluten-induced gut disorder that manifests itself with a range of clinical symptoms in genetically susceptible subjects. Immune reaction to wheat, barley and rye gliadin fractions and glutenins [1] triggers an inflammatory state of the duodenal mucosa: the result is reduced intestinal villus height and hyperplastic cryptae that may lead to complete villus atrophy. The critical role played by gluten is demonstrated by the fact that in CD patients on a gluten free diet (GFD) clinical symptoms disappear,
⁎ Corresponding author. Tel.: +39 0433 488261; fax: +39 0433 488697. E-mail addresses: [email protected] (E. Tonutti), [email protected] (N. Bizzaro). 1568-9972/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.autrev.2014.01.043
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
472 473 473 473 474 474 474 476
anti-transglutaminase 2 antibodies (anti-tTG2, the serological markers of the disorder) normalize, and villus atrophy recedes. As to the role of genetic factors, CD development has been demonstrated to be closely associated with MHC class II HLA-DQ2 and HLA-DQ8 molecules; in fact, virtually all CD patients express at least one of these HLA molecules compared to the general population in which about 30–35% have either DQ2 or DQ8 [2,3]. A new gluten-associated clinical condition, named ‘non-celiac gluten sensitivity’ (NCGS) [4], also described in literature as gluten hypersensitivity or gluten intolerance, has been recently identified. NCGS is characterized by gastrointestinal or extraintestinal symptoms comparable, in many cases, to those of CD patients; however, to date no specific immunological mechanisms or serological markers have been identified for this disorder. The diagnosis is made by exclusion of CD or IgE-mediated allergy to wheat, and is based on the direct association between gluten ingestion and symptom onset.
E. Tonutti, N. Bizzaro / Autoimmunity Reviews 13 (2014) 472–476
2. Pathogenesis CD is initiated by the ingestion of gliadin which is present in foods containing wheat, barley and rye. It is well established that the pathogenic mechanism is related to an alteration in the integrity of the tight junction system of intestinal epithelium cells, enabling the passage of macromolecules such as gluten in the submucosa. Gluten is an excellent substrate for transglutaminase 2 (TG2, also known as tissue transglutaminase). This enzyme converts glutamine residues into negatively charged glutamate residues in a process termed deamidation, thus facilitating the binding of gliadin peptides to HLA antigens of class II DQ2 or DQ8 expressed on antigen presenting cells. HLA DQ2or DQ8-restricted CD4 T cells are critical to the pathogenesis of CD. These cells recognize gluten selectively in the context of HLA-DQ2 or DQ8 molecules and are present only in the small intestinal mucosa of individuals with CD. A high number of cytotoxic intraepithelial T lymphocytes (IELs) that express activating NK cell receptors are also present in these patients. These cells, which are responsible for intestinal mucosal damage, are found in higher numbers in the presence of villus atrophy. CD patients develop autoantibodies against TG2 as well as to gluten (antibodies against deamidated gliadin peptides — DGP) suggesting that CD has an autoimmune component [5]. The relationship between CD and autoimmunity is further supported by epidemiological studies that show a link between CD and autoimmune disorders as type I diabetes and autoimmune thyroiditis [6]. Unlike CD, patients with NCGS are not affected by alterations of mucosal tight junctions or cytokine increases; conversely, there is evidence of overexpression of Toll-like receptor 2, a marker of innate immunity activation. It has therefore been proposed that NCGS and CD are different clinical syndromes and that NCGS may be associated with gluteninduced activation of innate, rather than adaptive, immune response [7]. 3. Epidemiology The development of highly sensitive immunological methods for identifying diagnostic antibodies (e.g. anti-tTG autoantibodies and anti-DGP antibodies) has enabled an increasing number of CD patients with vague or asymptomatic clinical presentations to be identified. Population-based studies now indicate that approximately 0.5–1% of the Western European and Northern American populations suffer from CD. In a recent paper, Abadie and coworkers [8] correlate gluten consumption with HLA DQ2 and DQ8 haplotype frequency in the populations of the different world countries. The authors found a significant correlation between CD prevalence and wheat consumption, and between CD prevalence and DQ2–DQ8 frequency in most countries. However, outlier countries have been observed: Finland and Russia, for example, have similar wheat consumption levels and comparable HLA haplotype frequencies, but the prevalence of CD in Finland is 1–2.4% whereas in the adjacent Russian republic of Karelia the prevalence of CD is considerably lower (0.2%). In the Maghreb area, wheat and barley are the major staple foods. Despite similar frequencies of the DR3–DQ2 and DR4–DQ8 haplotypes, the prevalence of CD in Algeria (5.6%) is by far the highest reported worldwide, whereas CD prevalence in Tunisia (0.28%) remains one of the lowest. These observations suggest that similar levels of wheat consumption and predisposing HLA expression can be associated with strikingly different levels of CD prevalence, which suggests the role of environmental factors and other genetic factors in CD pathogenesis. NGCS epidemiological data are too scarce to provide positive information. Fasano's team [4] reports a 6% prevalence in a population of patients who presented to Maryland gastroenterology clinics; this finding obviously does not refers to the general population, but to a selected population with clinical problems of a gastrointestinal nature. Bisiekierski [9] recently demonstrated, through a double blind study, that 30–40% of patients with inflammatory bowel syndrome diagnosed
473
by Rome III criteria are NCGS patients, as just one week after reintroduction of gluten into their diet they experience abdominal distension and pain.
4. Clinical manifestations CD is characterized by multiple clinical expressions. An ESPGHAN (European Society for Paediatric Gastroenterology, Hepatology and Nutrition) working group has recently developed new guidelines for the diagnosis of CD based on scientific and technical developments using an evidence-based approach [10]. The ESPGHAN working group decided to revise the classification, also taking into consideration signs and symptoms that had not been considered in the previous classification. In particular, it was deemed advisable to eliminate the distinction between classic and atypical CD based on symptoms, as atypical signs and symptoms (e.g. anemia, neuropathy, reduced bone density) may be considerably more common than classic symptoms (e.g. chronic diarrhea). Table 1 provides an extensive list of symptoms and signs of CD in children and adolescents. Conversely, the definitions of silent, latent and potential CD are still valid. Silent CD is defined as presence of positive serology (anti-tTG IgA or IgG in IgA deficient subjects), of HLA alleles compatible with CD, and of histological alterations typical of CD, in patients not affected with CD symptoms. Latent CD is defined as presence of compatible HLA without gastrointestinal symptoms in patients who have had a gluten-dependent enteropathy at some point of their life. Potential CD is defined as the presence of CD-specific antibodies and compatible HLA, without histological abnormalities in duodenal biopsies [10]. Patients suffering from certain disorders (especially Hashimoto's thyroiditis, type I diabetes, IgA deficiency and Down's syndrome) have a higher risk of developing CD than the normal population. In these patients it is advisable to perform HLA DQ2/DQ8 and serological tests for CD even in the absence of symptoms. CD and NGCS cannot be distinguished clinically, since the symptoms experienced by NGCS patients are often seen in CD. The definition of NGCS is a gluten reaction in which both allergic and autoimmune mechanisms have been ruled out (diagnosis by exclusion criteria). Specifically: symptoms similar to those of celiac or allergic patients must be present; in vivo and in vitro wheat allergy tests (prick test and specific IgE), as well as anti-tTG and EMA antibodies must be negative; duodenal histology must be normal; the patients must also experience a disappearance of the symptoms when on a GFD and their reappearance after the reintroduction of gluten. The most frequent symptoms in NGCS patients are abdominal pain, eczema or rash, headache, blurred vision, fatigue, diarrhea, depression, anemia, numbness in the legs, arms or fingers, and joint pain (Table 2).
Table 1 Signs and symptoms of CD in children and adolescents. Iron-deficiency anemia Other or unspecified anemia Anorexia Weight loss Abdominal distension/bloating Abdominal pain Vomiting Flatulence Diarrhea Short stature Growth failure Irritability Increase level of liver enzymes Chronic fatigue Failure to thrive Constipation Irregular bowel habits
474
E. Tonutti, N. Bizzaro / Autoimmunity Reviews 13 (2014) 472–476
Table 2 Signs and symptoms of patients with non celiac gluten sensitivity (NCGS). Abdominal pain Abdominal distension/bloating Diarrhea Eczema Rash Headache Foggy mind Fatigue Depression Anemia Numbness in the legs, arms Joint pain
Table 3 Algorithm proposed by the ESPGHAN to diagnose CD in symptomatic children (modified).
ad
suggestive
An An An
(CD, celiac disease; EMA, endomysial antibodies; tTG, tissue transglutaminase).
5. Antibodies The serological diagnosis of CD is based on the detection of class IgA anti-tTG and EMA antibodies. In patients with IgA deficiency, IgG antiDGP or anti-tTG is used [11]. Anti-tTG measurement can be performed on serum or plasma samples through different immunometric methods (ELISA, radioimmunoassay, fluorimetric assays, etc.) using human or recombinant purified TG2. Anti-tTG tests currently in use provide quantitative results expressed in arbitrary units and calculated on the basis of test-specific reference standards. The test to detect anti-EMAs requires fluorescent microscopy, and is subject to significant variability of interpretation. Despite these limits, EMA specificity is very high (98–100%), so much that this test is considered specific for a diagnosis of CD [12]. Tests for the detection of anti-DGP IgA and IgG are much more specific than native gliadin-based tests (anti-gliadin antibodies — AGA); in particular, the assay to detect anti-DGP antibodies of the IgG class is able to identify CD patients aged less than 2 or 3, who may test negative to IgA anti-tTG testing [13]. Therefore, IgA anti-tTG plus IgG anti-DGP seems to be the best test combination to diagnose CD [14,15]. On the other hand, cases have been described of low anti-tTG concentrations in disorders other than CD (other autoimmune pathologies, cancer, liver and infectious diseases). In general, these positive results are not associated with positive EMA: this explains the higher specificity of EMA compared to anti-tTG. Cases have been reported of anti-tTG and EMA IgA positive, asymptomatic pediatric patients (with CD-compatible HLA and negative histology), who become sero-negative over time (1 or 2 years). These are patients with potential CD. Therefore, in symptomless children with a positive celiac serology, the decision of performing an intestinal biopsy should be preceded by a period of repeated serological testing [16]. An important aspect, confirmed by numerous studies, is the correlation between anti-tTG count and histological damage. One of the latest studies assessed retrospectively 412 consecutive anti-tTG and EMA patients who received a biopsy for suspected CD: the subjects whose levels of anti-tTG were greater than 7-fold the cut-off value had a 99.7% positive predictive value for histological damage (with Marsh score N2) [17]. To date, there are no specific laboratory markers for NCGS; a recent study by Volta found that 78 patients with NCGS were AGA IgG positive in 56.4% of the cases and AGA IgA positive in 7.7% of the cases. All patients were negative for anti-DGP IgG and IgA, as well as for anti-tTG and EMA [18].
architectural changes in the mucosa and outlines three categories of CD-associated lesion: 1) Type 1: infiltrative 2) Type 2: infiltrative–hyperplastic 3) Type 3: atrophic: 3a) mild villous atrophy, 3b) moderate villous atrophy, 3c) total villous atrophy Analysis of multiple biopsies is important: patchiness of the lesion has been reported and recent work suggests that different degrees of severity may be present, even in the same bioptic fragment. The biopsies should be taken from the second/third portion of the duodenum and at least one biopsy should be taken from the duodenal bulb. Patients with NCGS do not exhibit significant alterations of the duodenal mucosa; histological negativity is an essential parameter for a diagnosis of NCGS. 7. Diagnostic criteria The diagnostic criteria proposed by ESPGHAN in 1990 [20] envisaged the performance of gastroduodenoscopy and histological confirmation of mucosal damage as the conclusive phase of the diagnostic process. These criteria did not indicate which serological tests should be positive, were not applicable to children aged below 2 years, and in any case required other clinical conditions to be ruled out. Therefore, in 2010, the ESPGHAN working group deemed appropriate to set out new criteria based on new knowledge and diagnostic tools developed in the last few years [10] (Table 3).
Table 4 Algorithm proposed by the ESPGHAN to diagnose CD in asymptomatic subjects at risk for CD (modified).
Po An An
6. Intestinal biopsy CD is characterized by varying degrees of atrophy of the intestinal mucosa, with reduced height or disappearance of the villi. The degree of the intestinal lesion is defined on the basis of the widely used Marsh–Oberhuber classification [19] which describes the degree of
Bi
(CD, celiac disease; EMA, endomysial antibodies; tTG, tissue transglutaminase).
E. Tonutti, N. Bizzaro / Autoimmunity Reviews 13 (2014) 472–476 Table 5 Criteria for the diagnosis of non celiac gluten sensitivity. • Negative immuno-allergy tests to wheat • Negative CD serology (EMA and/or anti-tTG) and no IgA deficiency • Negative duodenal histopathology • Presence of biomarkers of gluten immune-reaction (AGA) • Presence of clinical symptoms that can overlap with CD or wheat allergy • Resolution of the symptoms following a GFD • Positive gluten challenge (CD, celiac disease; EMA, endomysial antibodies; tTG, tissue transglutaminase; AGA, antigliadin antibodies; GFD, gluten-free diet).
The new CD guidelines are revolutionary in two major respects: the crucial role of serological tests in the diagnostic process of symptomatic subjects, and the detection of HLA DQ2/DQ8 in diagnosing asymptomatic subjects belonging to groups at risk of CD. Concerning the diagnosis of children and adolescents with signs and symptoms suggestive of CD, the ESPGHAN guidelines (Table 4) recommend, as the initial approach to symptomatic patients, testing for antitTG IgA antibodies as well as for total serum IgA to exclude IgA deficiency. As an alternative to total serum IgA, direct testing for IgG anti-DGP antibodies can be performed. The decision to perform IgA anti-tTG as the initial test in this population is based on the high sensitivity and specificity of the test, its widespread availability, and low costs compared with the EMA IgA test. A fundamental aspect of the new guidelines concerns the possibility of not necessarily performing an intestinal biopsy if the anti-tTG antibody levels are very high, as in these cases the specificity of the antibody is absolute. Indeed, pediatric gastroenterologists should discuss with the parents and the patient who is positive for anti-tTG antibody levels N10 times ULN (as appropriate for age) the option of omitting the biopsies and the implications of doing so. If the parents (patient) accept this option, then blood should be drawn for HLA and EMA testing. Because EMA testing depends on the quality and experience of the laboratory, the clinician must collaborate with a laboratory with documented
experience and high standards in immunohistochemistry. If the patient tests positive for EMA antibodies and positive for HLA-DQ2 or HLA-DQ8, then the diagnosis of CD is confirmed. A GFD is started and the patient is studied for improvement of symptoms and decline of antibodies. A later gluten challenge in these children is not required. On the other side, patients with positive anti-tTG antibody levels lower than 10 times the upper limit for the normal population (ULN) given by the manufacturer of this particular test should undergo upper endoscopy with multiple biopsies. On the other hand, patients with positive anti-tTG antibody levels lower than 10 times the upper limit for the normal population (ULN) given by the manufacturer of this particular test should undergo upper endoscopy with multiple biopsies. As far as diagnosis methods in asymptomatic pediatric patients belonging to at-risk groups are concerned, the ESPGHAN guidelines suggest a different procedure. In these patients, HLA-DQ2 and HLA-DQ8 testing as the initial action is probably cost-effective since a significant proportion of the patients can be excluded from further studies because they do not harbor DQ2 or DQ8 [21]. In individuals with DQ2 or DQ8 positivity, IgA anti-tTG and total serum IgA determination should be performed. If IgA anti-tTG is negative and IgA deficiency is excluded, then CD is unlikely; however, the disease may still develop later in life. Therefore, serological testing should be repeated at regular intervals. If anti-tTG antibodies are positive, then signs related to CD should be searched for (e.g. anemia, elevated liver enzymes). As to diagnosis of NCGS, current diagnostic criteria are shown in Table 5; as can be seen, most criteria are based on the exclusion of CD or wheat allergy (WA). Based on a combination of clinical, biological, genetic and histological data it is possible to differentiate between the three conditions (WA, CD and NCGS) following the algorithm shown in Fig. 1. In most cases, information coming from clinical presentation will be sufficient to distinguish WA. Detection of specific biomarkers for WA and CD is the first step in the diagnostic process. If these forms have been excluded, the NCGS should be considered. A double placebo gluten challenge will be the final step to confirm or rule out NCGS.
History and physical examination
Wheat allergy (WA)
Wheat skin prick test Wheat serum IgE (Gluten challenge)
Positive WA confirmed
Negative WA ruled out
475
Celiac disease (CD) Gluten sensitivity (GS) anti-tTG/EMA, total IgA, anti-deamidated gliadin peptides (DGP), anti-gliadin (AGA)
anti-tTG/EMA and/or anti-DGP pos
Gastroduodenoscopy and biopsy
Biopsy positive
CD diagnosis confirmed and gluten-free diet
anti-tTG/EMA and anti-DGP neg Gluten challenge
Gluten challenge positive
GS diagnosis confirmed
Fig. 1. Algorithm for the differential diagnosis of gluten-related disorders (modified by Sapone et al. [4]).
476
E. Tonutti, N. Bizzaro / Autoimmunity Reviews 13 (2014) 472–476
References [1] Shevry PR, Tatham AS, Kasarda DD. Cereal proteins and celiac disease. In: Marsh M, editor. Coeliac disease. Oxford, UK: Blackwell; 1992. p. 305–48. [2] Bevan S, Popat S, Braegger CP, Busch A, O'Donoghue D, Falth-Magnusson K, et al. Contribution of the MHC region to the familial risk of coeliac disease. J Med Genet 1999;36:687–90. [3] Hogberg L, Falth-Magnusson K, Grodzinsky E, Stenhammar L. Familial prevalence of celiac disease: a twenty-year follow-up study. Scand J Gastroenterol 2003;38:61–5. [4] Sapone A, Bai JC, Ciacci C, Dolinsek J, Green PH, Hadjivassiliou M, et al. Spectrum of gluten-related disorders: consensus of a new nomenclature and classification. BMC Med 2012;10:13. [5] Dieterich W, Ehnis T, Bauer M, Donner P, Volta U, Riecken EO, et al. Identification of tissue transglutaminase as the autoantigen of celiac dise ase. Nat Med 1997;3:797–801. [6] Ventura A, Magazzu G, Greco L. Duration of exposure to gluten and risk for autoimmune disorders in patients with celiac disease. SIGEP Study Group for autoimmune disorders in celiac disease. Gastroenterology 1999;117:297–303. [7] Sapone A, Lammers KM, Mazzarella G, Mikhailenko I, Cartenì M, Casolaro V, et al. Differential mucosal IL-17 expression in two gliadin-induced disorders: gluten sensitivity and the autoimmune enteropathy celiac disease. Int Arch Allergy Immunol 2010;152:75–80. [8] Abadie V, Sollid LM, Barreiro LB, Jabri B. Integration of genetic and immunological insights into a model of celiac disease pathogenesis. Annu Rev Immunol 2011;29:493–525. [9] Biesiekierski JR, Newnham ED, Irving PM, Barrett JS, Haines M, Doecke JD, et al. Gluten causes gastrointestinal symptoms in subjects without celiac disease: a doubleblind randomized placebo-controlled trial. Am J Gastroenterol 2011;106:508–14. [10] Husby S, Koletzko S, Korponay-Szabo IR, Mearin ML, Phillips A, Shamir R, et al. European Society for Paediatric Gastroenterology, Hepatology and Nutrition Guidelines for the diagnosis of celiac disease. J Paediatr Gastroenterol Nutr 2012;54:136–60.
[11] Villalta D, Tonutti E, Prause C, Koletzko S, Uhlig HH, Vermeersch P, et al. IgG antibodies against deamidated gliadin peptides for diagnosis of celiac disease in patients with IgA deficiency. Clin Chem 2010;56:464–8. [12] Lewis NR, Scott BB. Systematic review: the use of serology to exclude or diagnose celiac disease (a comparison of the endomysial and tissue transglutaminase antibody tests). Aliment Pharmacol Ther 2006;24:47–54. [13] Tonutti E, Visentini D, Picierno A, Bizzaro N, Villalta D, Tozzoli R, et al. Diagnostic efficacy of the ELISA test for the detection of deamidated anti-gliadin peptide antibodies in the diagnosis and monitoring of celiac disease. J Clin Lab Anal 2009;23:165–71. [14] Bizzaro N, Tozzoli R, Villalta D, Fabris M, Tonutti E. Cutting-edge issues in celiac disease and in gluten intolerance. Clin Rev Allergy Immunol 2010;42:279–87. [15] Brusca I, Carroccio A, Tonutti E, Villalta D, Tozzoli R, Barrale M, et al. The old and new tests for celiac disease: which is the best test combination to diagnose celiac disease in pediatric patients? Clin Chem Lab Med 2011;50:111–7. [16] Lionetti E, Castellaneta S, Pulvirenti A, Tonutti E, Francavilla R, Fasano A, et al. Prevalence and natural history of potential celiac disease in at-family-risk infants prospectively investigated from birth. J Pediatr 2012;161:908–14. [17] Alessio MG, Tonutti E, Brusca I, Radice A, Licini L, Sonzogni A, et al. Correlation between IgA tissue transglutaminase antibody ratio and histological finding in celiac disease. J Pediatr Gastroenterol Nutr 2012;55:44–9. [18] Volta U, Tovoli F, Cicola R, Parisi C, Fabbri A, Piscaglia M, et al. Serological tests in gluten sensitivity (nonceliac gluten intolerance). J Clin Gastroenterol 2012;46:680–5. [19] Oberhuber G, Granditsch G, Vogelsang H. The histopathology of celiac disease: time for a standardized report scheme for pathologists. Eur J Gastroenterol 1999;11:1185–94. [20] Walker-Smith JA, Guandalini S, Schmitz J. Revised criteria for diagnosis of celiac disease. Arch Dis Child 1990;65:909–11. [21] Csizmadia CG, Mearin ML, Oren A, Kromhout A, Crusius JB, von Blomberg BM, et al. Accuracy and cost-effectiveness of a new strategy to screen for celiac disease in children with Down syndrome. J Pediatr 2000;137:756–61.
Contents lists available at ScienceDirect
Autoimmunity Reviews journal homepage: www.elsevier.com/locate/autrev
Review
Diagnosis and classification of celiac disease and gluten sensitivity Elio Tonutti a, Nicola Bizzaro b,⁎ a b
Immunopathology and Allergology, Azienda Ospedaliero-Universitaria, Piazza S. Maria della Misericordia 15, 33100 Udine, Italy Laboratory of Clinical Pathology, San Antonio Hospital, via Morgagni 18, 33028 Tolmezzo, UD, Italy
a r t i c l e
i n f o
a b s t r a c t
Article history: Accepted 13 November 2013 Available online 15 January 2014
Celiac disease is a complex disorder, the development of which is controlled by a combination of genetic (HLA alleles) and environmental (gluten ingestion) factors. New diagnostic guidelines developed by ESPGHAN emphasize the crucial role of serological tests in the diagnostic process of symptomatic subjects, and of the detection of HLA DQ2/DQ8 alleles in defining a diagnosis in asymptomatic subjects belonging to at-risk groups. The serological diagnosis of CD is based on the detection of class IgA anti-tissue transglutaminase (anti-tTG) and antiendomysial antibodies. In patients with IgA deficiency, anti-tTG or anti-deamidated gliadin peptide antibody assays of the IgG class are used. When anti-tTG antibody levels are very high, antibody specificity is absolute and CD can be diagnosed without performing a duodenum biopsy. Non-celiac gluten sensitivity is a gluten reaction in which both allergic and autoimmune mechanisms have been ruled out. Diagnostic criteria include the presence of symptoms similar to those of celiac or allergic patients; negative allergological tests and absence of anti-tTG and EMA antibodies; normal duodenal histology; evidence of disappearance of the symptoms with a gluten-free diet; relapse of the symptoms when gluten is reintroduced. © 2014 Elsevier B.V. All rights reserved.
Keywords: Celiac disease Diagnostic criteria Gluten sensitivity Anti-tissue transglutaminase antibodies HLA DQ2/DQ8
Contents 1. Introduction . . . . . 2. Pathogenesis . . . . . 3. Epidemiology . . . . 4. Clinical manifestations 5. Antibodies . . . . . . 6. Intestinal biopsy . . . 7. Diagnostic criteria . . References . . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
1. Introduction Celiac disease (CD) is a chronic, immune-mediated, gluten-induced gut disorder that manifests itself with a range of clinical symptoms in genetically susceptible subjects. Immune reaction to wheat, barley and rye gliadin fractions and glutenins [1] triggers an inflammatory state of the duodenal mucosa: the result is reduced intestinal villus height and hyperplastic cryptae that may lead to complete villus atrophy. The critical role played by gluten is demonstrated by the fact that in CD patients on a gluten free diet (GFD) clinical symptoms disappear,
⁎ Corresponding author. Tel.: +39 0433 488261; fax: +39 0433 488697. E-mail addresses: [email protected] (E. Tonutti), [email protected] (N. Bizzaro). 1568-9972/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.autrev.2014.01.043
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
. . . . . . . .
472 473 473 473 474 474 474 476
anti-transglutaminase 2 antibodies (anti-tTG2, the serological markers of the disorder) normalize, and villus atrophy recedes. As to the role of genetic factors, CD development has been demonstrated to be closely associated with MHC class II HLA-DQ2 and HLA-DQ8 molecules; in fact, virtually all CD patients express at least one of these HLA molecules compared to the general population in which about 30–35% have either DQ2 or DQ8 [2,3]. A new gluten-associated clinical condition, named ‘non-celiac gluten sensitivity’ (NCGS) [4], also described in literature as gluten hypersensitivity or gluten intolerance, has been recently identified. NCGS is characterized by gastrointestinal or extraintestinal symptoms comparable, in many cases, to those of CD patients; however, to date no specific immunological mechanisms or serological markers have been identified for this disorder. The diagnosis is made by exclusion of CD or IgE-mediated allergy to wheat, and is based on the direct association between gluten ingestion and symptom onset.
E. Tonutti, N. Bizzaro / Autoimmunity Reviews 13 (2014) 472–476
2. Pathogenesis CD is initiated by the ingestion of gliadin which is present in foods containing wheat, barley and rye. It is well established that the pathogenic mechanism is related to an alteration in the integrity of the tight junction system of intestinal epithelium cells, enabling the passage of macromolecules such as gluten in the submucosa. Gluten is an excellent substrate for transglutaminase 2 (TG2, also known as tissue transglutaminase). This enzyme converts glutamine residues into negatively charged glutamate residues in a process termed deamidation, thus facilitating the binding of gliadin peptides to HLA antigens of class II DQ2 or DQ8 expressed on antigen presenting cells. HLA DQ2or DQ8-restricted CD4 T cells are critical to the pathogenesis of CD. These cells recognize gluten selectively in the context of HLA-DQ2 or DQ8 molecules and are present only in the small intestinal mucosa of individuals with CD. A high number of cytotoxic intraepithelial T lymphocytes (IELs) that express activating NK cell receptors are also present in these patients. These cells, which are responsible for intestinal mucosal damage, are found in higher numbers in the presence of villus atrophy. CD patients develop autoantibodies against TG2 as well as to gluten (antibodies against deamidated gliadin peptides — DGP) suggesting that CD has an autoimmune component [5]. The relationship between CD and autoimmunity is further supported by epidemiological studies that show a link between CD and autoimmune disorders as type I diabetes and autoimmune thyroiditis [6]. Unlike CD, patients with NCGS are not affected by alterations of mucosal tight junctions or cytokine increases; conversely, there is evidence of overexpression of Toll-like receptor 2, a marker of innate immunity activation. It has therefore been proposed that NCGS and CD are different clinical syndromes and that NCGS may be associated with gluteninduced activation of innate, rather than adaptive, immune response [7]. 3. Epidemiology The development of highly sensitive immunological methods for identifying diagnostic antibodies (e.g. anti-tTG autoantibodies and anti-DGP antibodies) has enabled an increasing number of CD patients with vague or asymptomatic clinical presentations to be identified. Population-based studies now indicate that approximately 0.5–1% of the Western European and Northern American populations suffer from CD. In a recent paper, Abadie and coworkers [8] correlate gluten consumption with HLA DQ2 and DQ8 haplotype frequency in the populations of the different world countries. The authors found a significant correlation between CD prevalence and wheat consumption, and between CD prevalence and DQ2–DQ8 frequency in most countries. However, outlier countries have been observed: Finland and Russia, for example, have similar wheat consumption levels and comparable HLA haplotype frequencies, but the prevalence of CD in Finland is 1–2.4% whereas in the adjacent Russian republic of Karelia the prevalence of CD is considerably lower (0.2%). In the Maghreb area, wheat and barley are the major staple foods. Despite similar frequencies of the DR3–DQ2 and DR4–DQ8 haplotypes, the prevalence of CD in Algeria (5.6%) is by far the highest reported worldwide, whereas CD prevalence in Tunisia (0.28%) remains one of the lowest. These observations suggest that similar levels of wheat consumption and predisposing HLA expression can be associated with strikingly different levels of CD prevalence, which suggests the role of environmental factors and other genetic factors in CD pathogenesis. NGCS epidemiological data are too scarce to provide positive information. Fasano's team [4] reports a 6% prevalence in a population of patients who presented to Maryland gastroenterology clinics; this finding obviously does not refers to the general population, but to a selected population with clinical problems of a gastrointestinal nature. Bisiekierski [9] recently demonstrated, through a double blind study, that 30–40% of patients with inflammatory bowel syndrome diagnosed
473
by Rome III criteria are NCGS patients, as just one week after reintroduction of gluten into their diet they experience abdominal distension and pain.
4. Clinical manifestations CD is characterized by multiple clinical expressions. An ESPGHAN (European Society for Paediatric Gastroenterology, Hepatology and Nutrition) working group has recently developed new guidelines for the diagnosis of CD based on scientific and technical developments using an evidence-based approach [10]. The ESPGHAN working group decided to revise the classification, also taking into consideration signs and symptoms that had not been considered in the previous classification. In particular, it was deemed advisable to eliminate the distinction between classic and atypical CD based on symptoms, as atypical signs and symptoms (e.g. anemia, neuropathy, reduced bone density) may be considerably more common than classic symptoms (e.g. chronic diarrhea). Table 1 provides an extensive list of symptoms and signs of CD in children and adolescents. Conversely, the definitions of silent, latent and potential CD are still valid. Silent CD is defined as presence of positive serology (anti-tTG IgA or IgG in IgA deficient subjects), of HLA alleles compatible with CD, and of histological alterations typical of CD, in patients not affected with CD symptoms. Latent CD is defined as presence of compatible HLA without gastrointestinal symptoms in patients who have had a gluten-dependent enteropathy at some point of their life. Potential CD is defined as the presence of CD-specific antibodies and compatible HLA, without histological abnormalities in duodenal biopsies [10]. Patients suffering from certain disorders (especially Hashimoto's thyroiditis, type I diabetes, IgA deficiency and Down's syndrome) have a higher risk of developing CD than the normal population. In these patients it is advisable to perform HLA DQ2/DQ8 and serological tests for CD even in the absence of symptoms. CD and NGCS cannot be distinguished clinically, since the symptoms experienced by NGCS patients are often seen in CD. The definition of NGCS is a gluten reaction in which both allergic and autoimmune mechanisms have been ruled out (diagnosis by exclusion criteria). Specifically: symptoms similar to those of celiac or allergic patients must be present; in vivo and in vitro wheat allergy tests (prick test and specific IgE), as well as anti-tTG and EMA antibodies must be negative; duodenal histology must be normal; the patients must also experience a disappearance of the symptoms when on a GFD and their reappearance after the reintroduction of gluten. The most frequent symptoms in NGCS patients are abdominal pain, eczema or rash, headache, blurred vision, fatigue, diarrhea, depression, anemia, numbness in the legs, arms or fingers, and joint pain (Table 2).
Table 1 Signs and symptoms of CD in children and adolescents. Iron-deficiency anemia Other or unspecified anemia Anorexia Weight loss Abdominal distension/bloating Abdominal pain Vomiting Flatulence Diarrhea Short stature Growth failure Irritability Increase level of liver enzymes Chronic fatigue Failure to thrive Constipation Irregular bowel habits
474
E. Tonutti, N. Bizzaro / Autoimmunity Reviews 13 (2014) 472–476
Table 2 Signs and symptoms of patients with non celiac gluten sensitivity (NCGS). Abdominal pain Abdominal distension/bloating Diarrhea Eczema Rash Headache Foggy mind Fatigue Depression Anemia Numbness in the legs, arms Joint pain
Table 3 Algorithm proposed by the ESPGHAN to diagnose CD in symptomatic children (modified).
ad
suggestive
An An An
(CD, celiac disease; EMA, endomysial antibodies; tTG, tissue transglutaminase).
5. Antibodies The serological diagnosis of CD is based on the detection of class IgA anti-tTG and EMA antibodies. In patients with IgA deficiency, IgG antiDGP or anti-tTG is used [11]. Anti-tTG measurement can be performed on serum or plasma samples through different immunometric methods (ELISA, radioimmunoassay, fluorimetric assays, etc.) using human or recombinant purified TG2. Anti-tTG tests currently in use provide quantitative results expressed in arbitrary units and calculated on the basis of test-specific reference standards. The test to detect anti-EMAs requires fluorescent microscopy, and is subject to significant variability of interpretation. Despite these limits, EMA specificity is very high (98–100%), so much that this test is considered specific for a diagnosis of CD [12]. Tests for the detection of anti-DGP IgA and IgG are much more specific than native gliadin-based tests (anti-gliadin antibodies — AGA); in particular, the assay to detect anti-DGP antibodies of the IgG class is able to identify CD patients aged less than 2 or 3, who may test negative to IgA anti-tTG testing [13]. Therefore, IgA anti-tTG plus IgG anti-DGP seems to be the best test combination to diagnose CD [14,15]. On the other hand, cases have been described of low anti-tTG concentrations in disorders other than CD (other autoimmune pathologies, cancer, liver and infectious diseases). In general, these positive results are not associated with positive EMA: this explains the higher specificity of EMA compared to anti-tTG. Cases have been reported of anti-tTG and EMA IgA positive, asymptomatic pediatric patients (with CD-compatible HLA and negative histology), who become sero-negative over time (1 or 2 years). These are patients with potential CD. Therefore, in symptomless children with a positive celiac serology, the decision of performing an intestinal biopsy should be preceded by a period of repeated serological testing [16]. An important aspect, confirmed by numerous studies, is the correlation between anti-tTG count and histological damage. One of the latest studies assessed retrospectively 412 consecutive anti-tTG and EMA patients who received a biopsy for suspected CD: the subjects whose levels of anti-tTG were greater than 7-fold the cut-off value had a 99.7% positive predictive value for histological damage (with Marsh score N2) [17]. To date, there are no specific laboratory markers for NCGS; a recent study by Volta found that 78 patients with NCGS were AGA IgG positive in 56.4% of the cases and AGA IgA positive in 7.7% of the cases. All patients were negative for anti-DGP IgG and IgA, as well as for anti-tTG and EMA [18].
architectural changes in the mucosa and outlines three categories of CD-associated lesion: 1) Type 1: infiltrative 2) Type 2: infiltrative–hyperplastic 3) Type 3: atrophic: 3a) mild villous atrophy, 3b) moderate villous atrophy, 3c) total villous atrophy Analysis of multiple biopsies is important: patchiness of the lesion has been reported and recent work suggests that different degrees of severity may be present, even in the same bioptic fragment. The biopsies should be taken from the second/third portion of the duodenum and at least one biopsy should be taken from the duodenal bulb. Patients with NCGS do not exhibit significant alterations of the duodenal mucosa; histological negativity is an essential parameter for a diagnosis of NCGS. 7. Diagnostic criteria The diagnostic criteria proposed by ESPGHAN in 1990 [20] envisaged the performance of gastroduodenoscopy and histological confirmation of mucosal damage as the conclusive phase of the diagnostic process. These criteria did not indicate which serological tests should be positive, were not applicable to children aged below 2 years, and in any case required other clinical conditions to be ruled out. Therefore, in 2010, the ESPGHAN working group deemed appropriate to set out new criteria based on new knowledge and diagnostic tools developed in the last few years [10] (Table 3).
Table 4 Algorithm proposed by the ESPGHAN to diagnose CD in asymptomatic subjects at risk for CD (modified).
Po An An
6. Intestinal biopsy CD is characterized by varying degrees of atrophy of the intestinal mucosa, with reduced height or disappearance of the villi. The degree of the intestinal lesion is defined on the basis of the widely used Marsh–Oberhuber classification [19] which describes the degree of
Bi
(CD, celiac disease; EMA, endomysial antibodies; tTG, tissue transglutaminase).
E. Tonutti, N. Bizzaro / Autoimmunity Reviews 13 (2014) 472–476 Table 5 Criteria for the diagnosis of non celiac gluten sensitivity. • Negative immuno-allergy tests to wheat • Negative CD serology (EMA and/or anti-tTG) and no IgA deficiency • Negative duodenal histopathology • Presence of biomarkers of gluten immune-reaction (AGA) • Presence of clinical symptoms that can overlap with CD or wheat allergy • Resolution of the symptoms following a GFD • Positive gluten challenge (CD, celiac disease; EMA, endomysial antibodies; tTG, tissue transglutaminase; AGA, antigliadin antibodies; GFD, gluten-free diet).
The new CD guidelines are revolutionary in two major respects: the crucial role of serological tests in the diagnostic process of symptomatic subjects, and the detection of HLA DQ2/DQ8 in diagnosing asymptomatic subjects belonging to groups at risk of CD. Concerning the diagnosis of children and adolescents with signs and symptoms suggestive of CD, the ESPGHAN guidelines (Table 4) recommend, as the initial approach to symptomatic patients, testing for antitTG IgA antibodies as well as for total serum IgA to exclude IgA deficiency. As an alternative to total serum IgA, direct testing for IgG anti-DGP antibodies can be performed. The decision to perform IgA anti-tTG as the initial test in this population is based on the high sensitivity and specificity of the test, its widespread availability, and low costs compared with the EMA IgA test. A fundamental aspect of the new guidelines concerns the possibility of not necessarily performing an intestinal biopsy if the anti-tTG antibody levels are very high, as in these cases the specificity of the antibody is absolute. Indeed, pediatric gastroenterologists should discuss with the parents and the patient who is positive for anti-tTG antibody levels N10 times ULN (as appropriate for age) the option of omitting the biopsies and the implications of doing so. If the parents (patient) accept this option, then blood should be drawn for HLA and EMA testing. Because EMA testing depends on the quality and experience of the laboratory, the clinician must collaborate with a laboratory with documented
experience and high standards in immunohistochemistry. If the patient tests positive for EMA antibodies and positive for HLA-DQ2 or HLA-DQ8, then the diagnosis of CD is confirmed. A GFD is started and the patient is studied for improvement of symptoms and decline of antibodies. A later gluten challenge in these children is not required. On the other side, patients with positive anti-tTG antibody levels lower than 10 times the upper limit for the normal population (ULN) given by the manufacturer of this particular test should undergo upper endoscopy with multiple biopsies. On the other hand, patients with positive anti-tTG antibody levels lower than 10 times the upper limit for the normal population (ULN) given by the manufacturer of this particular test should undergo upper endoscopy with multiple biopsies. As far as diagnosis methods in asymptomatic pediatric patients belonging to at-risk groups are concerned, the ESPGHAN guidelines suggest a different procedure. In these patients, HLA-DQ2 and HLA-DQ8 testing as the initial action is probably cost-effective since a significant proportion of the patients can be excluded from further studies because they do not harbor DQ2 or DQ8 [21]. In individuals with DQ2 or DQ8 positivity, IgA anti-tTG and total serum IgA determination should be performed. If IgA anti-tTG is negative and IgA deficiency is excluded, then CD is unlikely; however, the disease may still develop later in life. Therefore, serological testing should be repeated at regular intervals. If anti-tTG antibodies are positive, then signs related to CD should be searched for (e.g. anemia, elevated liver enzymes). As to diagnosis of NCGS, current diagnostic criteria are shown in Table 5; as can be seen, most criteria are based on the exclusion of CD or wheat allergy (WA). Based on a combination of clinical, biological, genetic and histological data it is possible to differentiate between the three conditions (WA, CD and NCGS) following the algorithm shown in Fig. 1. In most cases, information coming from clinical presentation will be sufficient to distinguish WA. Detection of specific biomarkers for WA and CD is the first step in the diagnostic process. If these forms have been excluded, the NCGS should be considered. A double placebo gluten challenge will be the final step to confirm or rule out NCGS.
History and physical examination
Wheat allergy (WA)
Wheat skin prick test Wheat serum IgE (Gluten challenge)
Positive WA confirmed
Negative WA ruled out
475
Celiac disease (CD) Gluten sensitivity (GS) anti-tTG/EMA, total IgA, anti-deamidated gliadin peptides (DGP), anti-gliadin (AGA)
anti-tTG/EMA and/or anti-DGP pos
Gastroduodenoscopy and biopsy
Biopsy positive
CD diagnosis confirmed and gluten-free diet
anti-tTG/EMA and anti-DGP neg Gluten challenge
Gluten challenge positive
GS diagnosis confirmed
Fig. 1. Algorithm for the differential diagnosis of gluten-related disorders (modified by Sapone et al. [4]).
476
E. Tonutti, N. Bizzaro / Autoimmunity Reviews 13 (2014) 472–476
References [1] Shevry PR, Tatham AS, Kasarda DD. Cereal proteins and celiac disease. In: Marsh M, editor. Coeliac disease. Oxford, UK: Blackwell; 1992. p. 305–48. [2] Bevan S, Popat S, Braegger CP, Busch A, O'Donoghue D, Falth-Magnusson K, et al. Contribution of the MHC region to the familial risk of coeliac disease. J Med Genet 1999;36:687–90. [3] Hogberg L, Falth-Magnusson K, Grodzinsky E, Stenhammar L. Familial prevalence of celiac disease: a twenty-year follow-up study. Scand J Gastroenterol 2003;38:61–5. [4] Sapone A, Bai JC, Ciacci C, Dolinsek J, Green PH, Hadjivassiliou M, et al. Spectrum of gluten-related disorders: consensus of a new nomenclature and classification. BMC Med 2012;10:13. [5] Dieterich W, Ehnis T, Bauer M, Donner P, Volta U, Riecken EO, et al. Identification of tissue transglutaminase as the autoantigen of celiac dise ase. Nat Med 1997;3:797–801. [6] Ventura A, Magazzu G, Greco L. Duration of exposure to gluten and risk for autoimmune disorders in patients with celiac disease. SIGEP Study Group for autoimmune disorders in celiac disease. Gastroenterology 1999;117:297–303. [7] Sapone A, Lammers KM, Mazzarella G, Mikhailenko I, Cartenì M, Casolaro V, et al. Differential mucosal IL-17 expression in two gliadin-induced disorders: gluten sensitivity and the autoimmune enteropathy celiac disease. Int Arch Allergy Immunol 2010;152:75–80. [8] Abadie V, Sollid LM, Barreiro LB, Jabri B. Integration of genetic and immunological insights into a model of celiac disease pathogenesis. Annu Rev Immunol 2011;29:493–525. [9] Biesiekierski JR, Newnham ED, Irving PM, Barrett JS, Haines M, Doecke JD, et al. Gluten causes gastrointestinal symptoms in subjects without celiac disease: a doubleblind randomized placebo-controlled trial. Am J Gastroenterol 2011;106:508–14. [10] Husby S, Koletzko S, Korponay-Szabo IR, Mearin ML, Phillips A, Shamir R, et al. European Society for Paediatric Gastroenterology, Hepatology and Nutrition Guidelines for the diagnosis of celiac disease. J Paediatr Gastroenterol Nutr 2012;54:136–60.
[11] Villalta D, Tonutti E, Prause C, Koletzko S, Uhlig HH, Vermeersch P, et al. IgG antibodies against deamidated gliadin peptides for diagnosis of celiac disease in patients with IgA deficiency. Clin Chem 2010;56:464–8. [12] Lewis NR, Scott BB. Systematic review: the use of serology to exclude or diagnose celiac disease (a comparison of the endomysial and tissue transglutaminase antibody tests). Aliment Pharmacol Ther 2006;24:47–54. [13] Tonutti E, Visentini D, Picierno A, Bizzaro N, Villalta D, Tozzoli R, et al. Diagnostic efficacy of the ELISA test for the detection of deamidated anti-gliadin peptide antibodies in the diagnosis and monitoring of celiac disease. J Clin Lab Anal 2009;23:165–71. [14] Bizzaro N, Tozzoli R, Villalta D, Fabris M, Tonutti E. Cutting-edge issues in celiac disease and in gluten intolerance. Clin Rev Allergy Immunol 2010;42:279–87. [15] Brusca I, Carroccio A, Tonutti E, Villalta D, Tozzoli R, Barrale M, et al. The old and new tests for celiac disease: which is the best test combination to diagnose celiac disease in pediatric patients? Clin Chem Lab Med 2011;50:111–7. [16] Lionetti E, Castellaneta S, Pulvirenti A, Tonutti E, Francavilla R, Fasano A, et al. Prevalence and natural history of potential celiac disease in at-family-risk infants prospectively investigated from birth. J Pediatr 2012;161:908–14. [17] Alessio MG, Tonutti E, Brusca I, Radice A, Licini L, Sonzogni A, et al. Correlation between IgA tissue transglutaminase antibody ratio and histological finding in celiac disease. J Pediatr Gastroenterol Nutr 2012;55:44–9. [18] Volta U, Tovoli F, Cicola R, Parisi C, Fabbri A, Piscaglia M, et al. Serological tests in gluten sensitivity (nonceliac gluten intolerance). J Clin Gastroenterol 2012;46:680–5. [19] Oberhuber G, Granditsch G, Vogelsang H. The histopathology of celiac disease: time for a standardized report scheme for pathologists. Eur J Gastroenterol 1999;11:1185–94. [20] Walker-Smith JA, Guandalini S, Schmitz J. Revised criteria for diagnosis of celiac disease. Arch Dis Child 1990;65:909–11. [21] Csizmadia CG, Mearin ML, Oren A, Kromhout A, Crusius JB, von Blomberg BM, et al. Accuracy and cost-effectiveness of a new strategy to screen for celiac disease in children with Down syndrome. J Pediatr 2000;137:756–61.