Resolving incomplete single nucleotide polymorphism tagging of HLA-DQ2.2 for coeliac disease genotyping using digital droplet PCR.

A hallmark of coeliac disease (CD) is the exceptionally strong genetic association with HLA-DQ2.5, DQ8, and DQ2.2. HLA typing provides information on CD risk important to both clinicians and researchers. A method that enables simple and fast detection of all CD risk genotypes is particularly desirable for the study of large populations. Single nucleotide polymorphism (SNP)-based HLA typing can detect the CD risk genotypes by detecting a combination of six SNPs but this approach can struggle to resolve HLA-DQ2.2, seen in 4% of European CD patients, because of the low resolution of one negatively predicting SNP. We sought to optimise SNP-based HLA typing by harnessing the additional resolution of digital droplet PCR to resolve HLA-DQ2.2. Here we test this two-step approach in an unselected sample of Mexican DNA and compare its accuracy to DNA typed using traditional exon detection. The addition of digital droplet PCR for samples requiring negative prediction of HLA-DQ2.2 enabled HLA-DQ2.2 to be accurately typed. This technique is a simple addition to a SNP-based typing strategy and enables comprehensive definition of all at-risk HLA genotypes in CD in a timely and cost-effective manner.

Parent-reported prevalence of food allergy in Mexican schoolchildren: A population-based study

BACKGROUND: Food allergy (FA) prevalence is well documented in developed countries and appears to be increasing, but remains unknown in most Latin American countries. We aimed to evaluate on a population basis the parent-reported prevalence of FA and its clinical characteristics in Mexican schoolchildren. METHODS: A validated Spanish version of a structured written questionnaire was administered to parents of schoolchildren aged 5-13 years old from Culiacan, Mexico. RESULTS: A total of 1049 parents responded to the survey (response rate, 84%). The estimated prevalence rates (95% CI) were: adverse food reactions 10.0% (8.3-11.9), 'perceived FA, ever' 5.5% (4.3-7.0), 'physician-diagnosed FA, ever' 4.9% (3.7-6.3), 'immediate-type FA, ever' 4.4% (3.3-5.8), 'mmediate-type FA, current' 3.5% (2.6-4.8), and anaphylaxis 1.2% (0.72-2.1). Immediate hypersensitivity reactions were mainly triggered by the consumption of shrimp (1.3%), other shellfish (0.7%), strawberry (0.6%), chocolate (0.5%), and egg (0.4%). Schoolchildren with 'immediate-type FA, current' had more atopic dermatitis and allergic rhinitis (p < 0.05), but not asthma or drug allergy (p > 0.05) than children without FA. All cases of anaphylaxis sought medical attention, but only one child had physician-diagnosed anaphylaxis and was advised to acquire an epinephrine autoinjector. CONCLUSIONS: The prevalence of 'immediate-type FA, current' to any food is 3.5% in Mexican schoolchildren. The poor recognition of anaphylaxis and the low frequency of prescription of epinephrine autoinjectors suggest that acute food-induced allergic reactions are not optimally managed in Mexico

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Parent-reported prevalence of food allergy in Mexican schoolchildren: A population-based study.

BACKGROUND: Food allergy (FA) prevalence is well documented in developed countries and appears to be increasing, but remains unknown in most Latin American countries. We aimed to evaluate on a population basis the parent-reported prevalence of FA and its clinical characteristics in Mexican schoolchildren. METHODS: A validated Spanish version of a structured written questionnaire was administered to parents of schoolchildren aged 5-13 years old from Culiacan, Mexico. RESULTS: A total of 1049 parents responded to the survey (response rate, 84%). The estimated prevalence rates (95% CI) were: adverse food reactions 10.0% (8.3-11.9), "perceived FA, ever" 5.5% (4.3-7.0), "physician-diagnosed FA, ever" 4.9% (3.7-6.3), "immediate-type FA, ever" 4.4% (3.3-5.8), "immediate-type FA, current" 3.5% (2.6-4.8), and anaphylaxis 1.2% (0.72-2.1). Immediate hypersensitivity reactions were mainly triggered by the consumption of shrimp (1.3%), other shellfish (0.7%), strawberry (0.6%), chocolate (0.5%), and egg (0.4%). Schoolchildren with "immediate-type FA, current" had more atopic dermatitis and allergic rhinitis (p<0.05), but not asthma or drug allergy (p>0.05) than children without FA. All cases of anaphylaxis sought medical attention, but only one child had physician-diagnosed anaphylaxis and was advised to acquire an epinephrine autoinjector. CONCLUSIONS: The prevalence of "immediate-type FA, current" to any food is 3.5% in Mexican schoolchildren. The poor recognition of anaphylaxis and the low frequency of prescription of epinephrine autoinjectors suggest that acute food-induced allergic reactions are not optimally managed in Mexico.

Assessing of Celiac Disease and Nonceliac Gluten Sensitivity.

The publication of papers on the topic of gluten related disorders has substantially increased over the last few years. This has motivated healthcare professionals to pay attention not only to celiac disease and wheat allergy but also to a condition termed nonceliac gluten sensitivity (NCGS). Until now this condition has been diagnosed clinically on the basis of exclusion criteria and clinical response to gluten withdrawal. In addition, recent research in this field has shown that other food components distinct from gluten are implicated in NCGS cases, thereby changing our general understanding of NCGS diagnosis in either individuals on gluten containing diets or those already following a gluten-free diet with no proper diagnostic work-up of celiac disease. With this in mind, the assessment of NCGS will require extensive knowledge of celiac disease manifestations and the laboratory tests commonly performed during diagnosis of celiac disease.

Ex-vivo whole blood secretion of interferon (IFN)-γ and IFN-γ-inducible protein-10 measured by enzyme-linked immunosorbent assay are as sensitive as IFN-γ enzyme-linked immunospot for the detection of gluten-reactive T cells in human leucocyte antigen (HLA)-DQ2·5(+) -associated coeliac disease.

T cell cytokine release assays are used to diagnose infectious diseases, but not autoimmune or allergic disease. Coeliac disease (CD) is a common T cell-mediated disease diagnosed by the presence of gluten-dependent intestinal inflammation and serology. Many patients cannot be diagnosed with CD because they reduce dietary gluten before medical workup. Oral gluten challenge in CD patients treated with gluten-free diet (GFD) mobilizes gluten-reactive T cells measurable by interferon (IFN)-γ enzyme-linked immunospot (ELISPOT) or major histocompatibility complex (MHC) class II tetramers. Immunodominant peptides are quite consistent in the 90% of patients who possess HLA-DQ2·5. We aimed to develop whole blood assays to detect gluten-specific T cells. Blood was collected before and after gluten challenge from GFD donors confirmed to have CD (n = 27, all HLA-DQ2·5(+) ), GFD donors confirmed not to have CD (n = 6 HLA-DQ2·5(+) , 11 HLA-DQ2·5(-) ) and donors with CD not following GFD (n = 4, all HLA-DQ2·5(+) ). Plasma IFN-γ and IFN-γ inducible protein-10 (IP-10) were measured by enzyme-linked immunosorbent assay (ELISA) after whole blood incubation with peptides or gliadin, and correlated with IFN-γ ELISPOT. No T cell assay could distinguish between CD patients and controls prior to gluten challenge, but after gluten challenge the whole blood IFN-γ ELISA and the ELISPOT were both 85% sensitive and 100% specific for HLA-DQ2·5(+) CD patients; the whole blood IP-10 ELISA was 94% sensitive and 100% specific. We conclude that whole blood cytokine release assays are sensitive and specific for detection of gluten-reactive T cells in CD; further clinical studies addressing the utility of these tests in patients with an uncertain diagnosis of CD is warranted.

Giardia lamblia infection induces different secretory and systemic antibody responses in mice.

The adult mouse model of Giardia lamblia infection serves as an excellent animal model to understand the immunological mechanisms involved in the control and clearance of Giardia infection. Little is known about the G. lamblia-specific antigens that stimulate the humoral immune response in this model of giardiasis. We analysed the secretory and systemic antibody responses to G. lamblia during primary and secondary infection in C3H/HeJ adult mice. Faecal IgA and Serum IgG anti-G. lamblia antibodies were observed at week 2 post-infection. Serum IgG responses remained constant over the next several weeks, whereas faecal IgA titres continued to rise from weeks 2-6 post-infection. Western blot analysis revealed that intestinal IgA and serum IgG antibody responses were directed toward several distinct proteins of G. lamblia. Certain proteins appeared to be recognized by both faecal IgA and serum IgG, whereas other antigens were specific for either the secretory or systemic antibody responses. G. lamblia primary and secondary infections were associated with differences in the antibody recognition pattern. The biochemical and immunological characterization of these antigens will help us to better understand the immunobiology of the G. lamblia-host interaction.