Valentina Baldas

Mass screening for coeliac disease using antihuman transglutaminase antibody assay

Aims: To determine coeliac disease prevalence by an anti-transglutaminase antibody assay in a large paediatric population; to evaluate acceptance of the screening programme, dietary compliance, and long term health effects. Methods: Cross-sectional survey of 3188 schoolchildren (aged 6–12) and prospective follow up of diagnosed cases. Main outcome measures were: prevalence of coeliac disease defined by intestinal biopsy or positivity to both human tissue transglutaminase and anti-endomysium antibodies in HLA DQ2-8 positive subjects; percentage of children whose families accepted screening; dietary compliance as defined by negativity for anti-transglutaminase antibodies; and presence of clinical or laboratory abnormalities at 24 month follow up. Results: The families of 3188/3665 children gave their consent (87%). Thirty biopsy proven coeliacs were identified (prevalence 1:106). Three other children testing positive for both coeliac related autoantibodies and HLA DQ2-8 but refusing biopsy were considered as having coeliac disease (prevalence 1:96). Of 33 cases, 12 had coeliac related symptoms. The 30 biopsy proven coeliacs followed a gluten-free diet. Of 28 subjects completing 18–24 months follow up, 20 (71.4%) were negative for anti-transglutaminase antibodies, while eight were slightly positive; symptoms resolved in all 12 symptomatic children. Conclusions: Prevalence of coeliac disease is high in Italian schoolchildren. Two thirds of cases were asymptomatic. Acceptance of the programme was good, as was dietary compliance. Given the high prevalence and possible complications of untreated coeliac disease, the availability of a valid screening method, and evidence of willingness to comply with dietary treatment population mass screening deserves careful consideration.

Development of a novel rapid non-invasive screening test for coeliac disease

BACKGROUND Coeliac disease is one of the commonest underdiagnosed diseases in general practice. The autoantigen recognised by the sera of patients with coeliac disease has recently been identified as tissue transglutaminase. AIMS We evaluated a simple non-invasive immunological dot blot assay for coeliac disease, suitable for use by the general physician in the ambulatory setting. The sensitivity and specificity of this dot blot assay based on recognition of recombinant human transglutaminase were compared with those of antiendomysial antibodies and an enzyme linked immunosorbent assay. METHODS Serum samples were analysed from 64 healthy controls, 58 first degree relatives of coeliacs, 74 diseased controls, and 70 biopsy confirmed untreated patients with coeliac disease. Dot blot assay and enzyme linked immunosorbent assay were performed using recombinant human transglutaminase as antigen. RESULTS The dot blot assay, which can be performed in 20 minutes, was positive in all 70 untreated coeliacs (sensitivity 100%). Among the three control groups, there were three false positive tests by dot blot (specificity 98%), all belonging to the group of healthy subjects. The antiendomysial antibodies test missed five untreated coeliac patients (sensitivity 93%) and was negative in all three control groups (specificity 100%). The specificity of the immunosorbent assay was 99% for IgA and 98% for IgG, while sensitivity was 93% for IgA, 47% for IgG, and 100% for IgA and IgG combined. CONCLUSIONS The dot blot assay is highly accurate in detecting untreated subjects with coeliac disease and can be performed in the general physicians medical office during the course of a routine examination. This innovative test is a practical, reliable alternative to both the immunofluorescent based antiendomysial test and immunosorbent assay for detection of transglutaminase antibodies for the diagnosis of coeliac disease.

Looking for celiac disease : Diagnostic accuracy of two rapid commercial assays

BACKGROUND:Early diagnosis and treatment with gluten-free diet reduces mortality and the prevalence of associated disorders in celiac disease (CD). A simple “in the office” test of anti-transglutaminase antibodies might be of great help in first-line screening for CD.AIMS:We evaluated the sensitivity and specificity of two commercial kits based, respectively, on rapid detection of IgA-IgG anti-human-transglutaminase antibodies (anti-h-tTG) in serum and IgA anti-h-tTG antibody in one drop of whole blood. These assays were compared to a well-established enzyme-linked immunosorbent assay technique.METHODS:Serum samples were analyzed from 114 biopsy-confirmed celiacs, 120 healthy controls, 20 first-degree relatives of celiacs, and 75 diseased controls. The whole blood samples were analyzed from 51 biopsy-confirmed celiacs and 100 controls.RESULTS:The serum-based test was positive in all 114 celiacs (sensitivity 100%). Among the controls there were seven healthy blood donors, one first-degree relative, and three diseased controls who tested positive (specificity 94.9%). The blood drop-based assay testing IgA antibodies was positive in 46 of 51 (sensitivity 90.2%), and since three of the five patients testing negative had total IgA deficiency, the sensitivity value can be increased to 95.8%. All 100 controls tested negative (specificity 100%).CONCLUSIONS:The commercial kits described here produce high values of sensitivity and specificity, offering the general practitioner who suspects a possible case of CD the real possibility to look for anti-h-tTG antibodies in his own medical office during a standard visit at a satisfyingly low cost.

Evidence of a correlation between mannose binding lectin and celiac disease: a model for other autoimmune diseases

Celiac disease is a multifactorial disorder caused, in genetically susceptible patients, by the ingestion of dietary gluten. Very little is known about the genetic factors, but there is a strong association of two HLA haplotypes (DQ2 or α1*05, β1*02 and DQ8 or α1*0301, β1*0302) with the disease. We investigated the relationship between polymorphisms in the first exon of the MBL2 gene, which encodes for mannose binding lectin (MBL) and celiac disease. Moreover we studied the MBL role by immunohistochemistry and TUNEL. Results were confirmed by clinical findings. We enrolled 149 Italian celiac patients; 116 were characterized by the presence of DQ2 or DQ8. The HLA haplotype was established by allelic specific PCR while the MBL2 genotype was resolved by melting temperature assay. Immunohistochemistry and TUNEL assays were performed on serial sections of biopsy specimens from celiac patients and healthy controls. MBL2 allele and genotype frequencies varied significantly between celiac patients and healthy controls. The frequencies of the 0 allele were 28% in DQ2 or DQ8 celiac patients, 36% in HLA atypical celiac patients, and 22% in healthy controls. Interestingly, the MBL2 0/0 genotype was present in 7 of 33 HLA atypical celiac patients (21%) and in 13 of 116 HLA typical celiac patients (13%) but in only 7 of 147 healthy controls (5%). Furthermore, we found that MBL2 genotype is strongly associated with the occurrence of secondary autoimmune diseases. Immunohistochemistry and TUNEL findings support a role of MBL2 in the clearance of apoptotic cells. In conclusion, MBL2 variants, responsible for lower MBL levels, are associated with celiac disease and higher risk of developing autoimmune diseases. Here we propose a role for MBL in the disease which could be easily applied to other autoimmune disorders.

A reliable screening procedure for coeliac disease in clinical practice.

Background: The main autoantigen recognized by the sera of patients with coeliac disease (CD) is tissue transglutaminase (tTG). A human-recombinant form of tTG was used to develop an ELISA to measure anti-tTG serum antibodies for the diagnosis of CD. Preliminary retrospective reports suggest that the human tTG-based ELISA could identify coeliac patients missed by the IgA-anti-endomysium antibody test (AEA). Whether the human recombinant tTG ELISA is sufficiently accurate to become the main diagnostic CD tool in everyday clinical practice is unknown. The objective was to determine, in a prospective study, the sensitivity and specificity of an ELISA test based on the use of human tTG compared with AEA, to analyse the discordant cases for HLA DQ2-8 and for clinical and intestinal biopsy characteristics. Methods: 1106 patients referred to a gastrointestinal outpatient clinic for symptoms attributable to CD, 52 first-degree relatives of CD patients and 200 healthy controls were tested for both anti-human tTG and AEA antibodies. Results: Out of 1158 subjects, 146 were tested positive for anti-tTG antibodies and 140 were biopsy-proven coeliacs. The AEA test identified 126/1158 coeliacs who also tested positive for anti-tTG antibodies. The 14 patients missed by the AEA test carried the typical HLA-DQ for CD; they had normal levels of total serum IgA and had milder pathology than those with both anti-tTG and AEA positivity ( P < 0001). Conclusions: These results prove that human tTG-based ELISA is an excellent diagnostic tool for CD, for mass screening by both the specialist and the general clinic.

Celiac disease in patients with sporadic and inherited cardiomyopathies and in their relatives

AIMS To investigate celiac disease (CD) and related co-morbidity in patients with familial and sporadic cardiomyopathy and in their relatives. METHODS AND RESULTS We screened anti-human-tissue-transglutaminase (IgA and IgG anti-h-tTG) and anti-endomysial antibodies (AEAs) in 238 consecutive adult patients with inherited or sporadic dilated cardiomyopathy (DCM), 418 relatives, and 2000 healthy blood donors. HLADQ2-DQ8 was tested in tTG-positive subjects. The IgA-tTG-positive patients with cardiomyopathy underwent duodenal biopsy. Twenty-six subjects were tTG-positive: five DCM patients (2.1%), two of 28 (7.1%) and three of 390 (0.7%) relatives with and without echocardiographic abnormalities respectively, and 16 controls (0.8%). Twenty-two of 26 subjects were AEA-positive, and 25 HLA-positive. Of the five patients with cardiomyopathy and biopsy-proven CD, four suffered iron-deficiency anaemia. Two CD-positive DCM patients and two tTG-positive relatives were from families with inherited disease in which CD did not co-segregate with DCM. CONCLUSIONS; The higher prevalence of CD in patients with sporadic or inherited DCM, and of tTG-positive serology in relatives with echocardiographic abnormalities, suggests that immune-mediated mechanisms are active in subsets of patients/families. However, gluten intolerance cannot be considered causative since CD seems to be associated but not co-segregated with DCM in familial cases.

Anti-brain but not celiac disease antibodies in Landau-Kleffner Syndrome and related epilepsies

The Landau-Kleffner syndrome, the continuous spikes-waves during slow sleep syndrome and the benign epilepsy of childhood with rolandic spikes are rare childhood epilepsies with unknown etiology. Improvement in patients treated with immunoglobulin suggests an involvement of the immune system. We provide immunohistochemical evidence of autoantibodies against rat brain auditory cortex, brainstem and cerebellum, in children suffering with one or more of these syndromes. Only 1/14 patient was celiac.

Variant mannose-binding lectin alleles are associated with celiac disease

Abstract. In this study, we investigated the role of mannose-binding lectin (MBL) in celiac disease, by performing genotype analysis for the three point mutations in the first exon of the gene in 117 Italian celiac patients (characterized by flat biopsy and positive for anti-endomysium antibody and human transglutaminase antibodies) and 130 pan-ethnic healthy controls. The frequency of homozygous mutant 0/0 was significantly higher in the 117 Italian celiac patients (0.13) than in the 130 pan-ethnic healthy controls (0.05; P=0.0405). An increased frequency of homozygous 0/0 allele was found among patients with celiac disease compared with controls. These results suggest an involvement of MBL in the pathophysiology of celiac disease.