Interpreting Tests for Coeliac Disease

CLINICAL

Interpreting tests for coeliac disease

Tips, pitfalls and updates

Jason A Tye-Din COELIAC DISEASE is an immune illness, coeliac disease serology, but this is triggered by dietary gluten, that causes a uncommon and excluding other causes of broad range of gastrointestinal and extra- villous atrophy (see below) is important. This article is the second in a series intestinal manifestations.1 Untreated Testing at-risk individuals is strongly on pathology testing. Articles in this disease reduces quality of life, increases recommended to detect cases before series aim to provide information about healthcare use and is associated with substantial morbidity develops.3,6 An emerging laboratory tests that general 2–4 practitioners (GPs) may encounter. substantial morbidity. Mortality is active case-finding approach can improve increased because of lymphoproliferative detection of coeliac disease by more than Background malignancy, sepsis and refractory 40-fold,7 but this only works when doctors Coeliac disease is one of most prevalent disease.3 As 1.5% of Australians have are mindful of the disease. Approximately autoimmune illnesses encountered coeliac disease, it is one of the most 30 at-risk individuals need to be tested in general practice, and GPs have a common autoimmune illnesses that to find a positive case of coeliac disease.7 central role in its diagnosis and follow- up. Key challenges are improving its general practitioners (GPs) will encounter. There is insufficient evidence to support poor rate of detection, distinguishing it However, its broad and often subtle population screening.8 Figure 2 provides from ‘gluten sensitivity’, and monitoring presentation makes detection challenging, an outline of a recommended diagnostic and optimising treatment to enhance and means 80% of Australians with pathway. long‑term outcomes. coeliac disease remain undetected.5 As Objective expeditious diagnosis and treatment with When to test for coeliac disease The objective of this article is to review a strict, lifelong gluten-free diet (GFD) the evidence-based use of serology, minimises long-term complications,3 Symptoms and clinical features that histology and genetic testing in the application of the appropriate tests to identify patients who might benefit diagnosis and follow-up care of adults ensure accurate diagnosis and follow-up from testing are shown in Table 1.9 and children with coeliac disease. is crucial. ‘Classical’ symptoms caused by intestinal Discussion inflammation, such as diarrhoea and Recognition and testing of at-risk Making the diagnosis weight loss, are frequent, but the ‘non- patients are keys to expediting classical’, extra-intestinal manifestations the diagnosis of coeliac disease. In clinical practice, suspected patients are even more common. These non- Knowing when and how to use are generally screened with coeliac classical features include lethargy, serology, histology, human leukocyte disease serology. In patients with headaches, osteoporosis, iron deficiency, antigen typing and gluten challenge positive coeliac disease serology, the transaminase elevation, infertility, other will increase the accuracy of both diagnosis and disease monitoring. diagnosis is confirmed by the presence autoimmune disease and dermatitis of characteristic small intestinal mucosal herpetiformis. A positive family history changes. The key diagnostic features are: of coeliac disease carries the strongest • intestinal histology showing raised predictive value for the disease. intraepithelial lymphocytes (>25 per 100 enterocytes), crypt hyperplasia and Tips and pitfalls villous atrophy (Figure 1) • Coeliac disease can develop at any age. • disease remission confirmed by The median age of diagnosis is 40 years, symptom resolution, normalised coeliac but do not discount coeliac disease in disease serology and, most reliably and the young and elderly. importantly, mucosal healing following • Coeliac disease affects both sexes, treatment with a GFD.3,6 with a modest female predominance. Correlation of histology and serology Men with coeliac disease are often with clinical history is important. Coeliac overlooked.5 disease can be present despite negative • Coeliac disease is a global disorder that

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is common in Western populations, Coeliac serology is typically performed with intravenous North Africa, the Middle East, India sedation, is simple and safe, and takes and Pakistan.10 Reports from Asian Currently, serologic testing for coeliac as little as 10 minutes. As coeliac disease countries, such as China, are on the rise. disease consists of the transglutaminase causes patchy involvement of the proximal Coeliac disease should not be excluded (tTG) and deamidated gliadin peptide small intestine, multiple biopsies are on the basis of a patient’s ethnicity or (DGP) antibody tests. In practice, both recommended (eg two from the first appearance. tests have >85% sensitivity and >90% and four from the second part of the • Clinical heterogeneity is substantial. specificity.12 The DGP assay has replaced duodenum).3,6 Endoscopic changes of Some patients have minimal or the whole-protein anti-gliadin antibody coeliac disease, such as mucosal scalloping, no obvious symptoms, or only (AGA) assay because of improved are occasionally seen, but diagnosis rests extra‑intestinal issues. One-third specificity; however, many labs will on the microscopic appearance. of patients with coeliac disease are report the DGP result as the ‘anti-gliadin overweight or obese at diagnosis.11 antibody’. The anti-endomysial antibody Tips and pitfalls (EMA) test measures tTG antibodies, but is • Villous atrophy is suggestive but labour-intensive, user-dependent and less not pathognomonic of coeliac widely performed. Testing approaches are disease. Other causes to consider, shown in Table 2. especially if coeliac disease serology is negative, include Giardia, common Tips and pitfalls variable immunodeficiency, Crohn’s • Positive coeliac disease serology in disease, tropical sprue, autoimmune isolation is insufficient for the diagnosis enteropathy, cow’s milk protein of coeliac disease. intolerance and some medications • The higher the titre of serology, the (eg olmesartan). greater the positive predictive value for • A GFD or immunosuppression can coeliac disease.13 obscure changes of villous atrophy. • Coeliac disease serology has a false • Correct biopsy processing and negative rate of 10–15%.14 Check if interpretation by a skilled pathologist your patient is on a GFD or taking is vital. When there is diagnostic immunosuppressants. uncertainty, review of the pathology • The tTG normal range varies can be informative. by manufacturer as there is no international standard. Comparing A Human leukocyte antigen titres is not possible if different labs or DQ2/8 genotyping tTG assays are used. • In patients with risk factors for coeliac The strong association between coeliac disease, negative coeliac disease disease and specific human leukocyte serology has lower negative predictive antigen (HLA) genes makes HLA value, so further work-up should be genotyping a useful tool in specific considered.15 situations (Table 3).16 The main • Point-of-care tests to detect coeliac susceptibility genes are HLA-DQ2 disease antibodies have not been (specifically HLA-DQ2.5 and HLA-DQ2.2) validated in primary practice, so cannot and HLA-DQ8, which are collectively currently be recommended.11 seen in almost all (99%) patients with • Patients with persistently positive coeliac disease, compared with 40–50% B coeliac disease serology but normal of the Australian community.5 Although small intestinal histology may have these genes, especially HLA-DQ2.5, Figure 1. Healthy small intestine, compared ‘potential’ (or ‘latent’) coeliac disease, impart substantial relative risk for coeliac with villous atrophy in coeliac disease. and follow-up is recommended.3,6 disease, the absolute risk is low, and most A. Normal small intestinal mucosa in adequately treated coeliac disease patients with one or more of these genes B. Untreated coeliac disease showing the Gastroscopy and small bowel will not develop coeliac disease. HLA-DQ7 classic triad of infiltration of the epithelium biopsies (composed of half the DQ2.5 allele, with lymphocytes, crypt hyperplasia and DQA1*05) may impart a very low risk for villous atrophy 16 Magnification ×100, haematoxylin and Histological evaluation of biopsies from coeliac disease but this remains unclear. eosin stain. the small intestine is the cornerstone of The main benefit of HLA typing is its coeliac disease diagnosis.3,6 Gastroscopy ability to exclude coeliac disease diagnosis

Option 1: HLA-DQ2/8 genotyping 9 Table 1. When to test for coeliac disease The absence of HLA susceptibility Offer serological testing for coeliac disease to people with any of the following: means that coeliac disease is unlikely and further investigations can focus on • Persistent unexplained abdominal or gastrointestinal symptoms other diagnoses. The presence of HLA • Faltering growth susceptibility genes is not diagnostic of • Prolonged fatigue coeliac disease, so option 2 is required. • Unexpected weight loss • Severe or persistent mouth ulcers Option 2: Gluten challenge, then testing • Unexplained iron, vitamin B12 or folate deficiency The amount and duration of gluten • Type 1 diabetes, at diagnosis required to consistently trigger diagnostic • Autoimmune thyroid disease, at diagnosis changes of coeliac disease appears highly • Irritable bowel syndrome (in adults) variable and more research is required. • First-degree relatives of people with coeliac disease Approximately 3–6 g of gluten consumed Consider serological testing for coeliac disease in people with any of the following: daily for two weeks will cause intestinal changes of coeliac disease in 50–70% of • Metabolic bone disorder (reduced bone mineral density or osteomalacia) affected adults, with the development • Unexplained neurological symptoms (particularly peripheral neuropathy or ataxia of positive serology after four weeks in • Unexplained subfertility or recurrent miscarriage 10–55%.19,20 To optimise the diagnostic • Persistently raised liver enzymes with unknown cause yield, patients should be encouraged to • Dental enamel defects return to consuming 3–6 g or more of gluten • Down syndrome each day for, ideally, six or more weeks. • Turner syndrome This daily amount of gluten can be found Reproduced with permission from the National Institute for Health and Care Excellence. Coeliac disease: Recognition, in two to four slices of wheat bread, two to assessment and management. London: NICE, 2015. Available at www.nice.org.uk/guidance/ng20 four Weet-Bix or 0.5–1 cup of cooked pasta.

Tips and pitfalls when the susceptibility genotypes are Gluten-sensitive or • Symptomatic relapse with a gluten absent (likelihood of coeliac disease <1%). wheat‑sensitive patients challenge is common, but has poor A positive HLA test does not diagnose predictive value for coeliac disease. coeliac disease, but indicates that Many Australians adopt a GFD without Gluten challenge is informative only if further investigation may be warranted. assessment for coeliac disease. This accompanied by objective testing. Genotyping is widely available through poses a diagnostic dilemma as coeliac • The tolerability of a gluten challenge commercial labs in Australia with a disease serology and intestinal histology may be improved by commencing with request for ‘HLA-DQ2/8 genotyping’. It can become falsely negative if the patient a small amount of gluten and slowly is performed on a blood sample, but can has been on a GFD for more than a increasing over subsequent days, and be done on a buccal scrape through some few months. Many Australians remove consuming it in divided doses over the collection centres (patients can check gluten from their diet because they feel course of the day (eg breakfast and in advance). HLA typing reports can be it helps improve gastrointestinal or other lunch). Fermented breads with lower difficult to interpret, so Australasian symptoms.17 For these people, a definitive FODMAP content (that still contain guidelines have been developed to simplify diagnosis is desirable as: gluten) are commercially available and and standardise reporting.16 • a formal diagnosis of coeliac disease will may also improve challenge tolerability. ensure strict treatment and follow-up of Tips and pitfalls a serious medical illness Family screening • HLA typing is expensive (Medicare • many people who self-report ‘gluten Benefits Schedule item number 71151; sensitivity’ are not actually sensitive The risk of coeliac disease in patients who $118.85), so it is important to use it to gluten. These patients, instead of have an affected family member with the prudently (Table 3).16 excluding gluten, may benefit more disease is 10%, but increases up to 20% • HLA typing is a ‘once only’ test as a from excluding other symptom-inducing if multiple family members are affected. person’s genotype does not change. wheat components, such as fermentable Screening patients with a family history of • HLA typing results are not adversely carbohydrates (FODMAPs).18 coeliac disease is important and strongly affected by a GFD. There are two diagnostic approaches – indicated when there are suggestive • Most patients with HLA susceptibility option 1 may be appropriate if the patient symptoms or signs.3,6 As it is increasingly for coeliac disease will not have the is unwilling to undertake the gluten recognised that many ‘asymptomatic’ disease or ever develop it. challenge; however, option 2 is definitive. patients with coeliac disease have

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Doctor considers coeliac disease (Table 1) Following a gluten-free diet

Option 2 Consuming Option 1: If initially gluten in diet unwilling to do gluten challenge

Coeliac serology HLA DQ2/8 genotyping Gluten (Table 2) (Box 1) challenge At-risk or Low-risk patient clinical suspicion

Positive tTG and/or DGP Coeliac disease unlikely

Specialist review May benefit from further Not coeliac Gastroscopy + small investigation such as HLA disease intestinal biopsies DQ2/8 genotyping and CD serology can also be performed gastroscopy and biopsies

Characteristic Normal coeliac histology histology Equivocal

Coeliac disease Specialist review Not coeliac disease Needs further investigation Note: If CD serology is such as review of pathology, consistently positive consider HLA DQ2/8 genotyping, and Follow-up to confirm ‘potential coeliac disease’ possibly gluten challenge and improvement on GFD repeat biopsies

Figure 2. Recommended diagnostic pathway for coeliac disease CD, coeliac disease; DGP, deamidated gliadin peptide antibody; tTG, tissue transglutaminase antibody

underlying nutrient deficiencies, disease. If HLA susceptibility is present the age of four years if they are well reduced bone density, or have symptom but coeliac disease serology is normal, the and symptom-free. improvement following a GFD (indicating individual is at risk for future development • Remind your patients with coeliac they were never asymptomatic), this of the disease. Repeat coeliac disease disease that their relatives are at means all relatives, irrespective of serology would be recommended if increased risk of the disease and symptom status, should be considered for they develop suggestive symptoms. If should be considered for testing. screening.21 First-degree relatives should asymptomatic, some experts recommend be screened, and if there are several screening every two to three years during Paediatric testing affected family members second-degree childhood to avoid the detrimental effects relatives should also be tested. of unrecognised coeliac disease on growth Although similar to adults, there are Family screening using HLA DQ2/8 and bone health.13 additional considerations when assessing genotyping with coeliac disease serology children for coeliac disease.22 New is more informative than serology alone.16 Tips and pitfalls European guidelines, based on evidence A relative without HLA susceptibility • Screening children with a family history that high-titre tTG is strongly predictive does not require monitoring for coeliac of coeliac disease can be delayed until of coeliac disease in children, suggest

small intestinal biopsies can be avoided Table 2. How to test for coeliac disease if children meet the following criteria:13 • characteristic symptoms of coeliac 1. Confirm your patient is consuming a normal, gluten-containing diet disease 2. Request coeliac serology as follows: • tTG-IgA levels >10× upper limit of Option 1: Transglutaminase-IgA (tTG-IgA) + Deamidated gliadin peptide-IgG (DGP-IgG) normal – Medicare Benefits Schedule (MBS) item number 71164, double antibody test • a positive endomysial antibody (EMA) ($39.90) is the preferred* one-step approach. on a different blood sample Or • positive HLA susceptibility for coeliac Option 2: Transglutaminase-IgA (tTG-IgA) + Total IgA level† – If the IgA level is low, disease. perform the deamidated gliadin peptide-IgG (DGP-IgG). MBS item number The utility of this approach in Australia 71163, single antibody test ($24.75). is uncertain because of the limited 3. If tTG-IgA and/or DGP-IgG is positive, irrespective of titre, refer for confirmatory small availability of the EMA test, as well intestinal biopsy as intra-lab variation and lack of standardisation of the tTG assay. Further *Option 1 overcomes the need to assess the total IgA level by performing DGP-IgG, which is not adversely affected by IgA deficiency. Further, DGP-IgG enhances the pick-up of coeliac disease by 15% compared to tTG-IgA alone.25 validation is warranted. The decision to Positive tTG-IgA and DGP-IgG together provides greater predictive value for coeliac disease than either alone.26 make a non-biopsy diagnosis should only †Total IgA level detects the 3% of people with coeliac disease with selective IgA deficiency that can cause false negative results. be made with specialist paediatric input.

Tips and pitfalls • The tTG assay has lower sensitivity in small dietary indiscretions, and intestinal • Distinguishing coeliac disease from children under three years of age. Ensure biopsies are recommended when disease ‘gluten sensitivity’ has important DGP-IgG testing is performed alongside activity needs to be accurately assessed. implications for the patient and their tTG-IgA to overcome this issue. family’s medical care. • Children with coeliac disease may Conclusion • Do not forget family screening given the present with more ‘classical’ complaints insidious nature and adverse outcomes than adults,22 but be mindful of extra- Coeliac disease is highly prevalent in of undiagnosed coeliac disease.26,27 intestinal issues. Anxiety, depression, general practice. Good patient care aggressive behaviour and sleep depends on knowing when and how to Authors 23 problems can be a presenting feature. test for it and how to monitor progress. Jason A Tye-Din MBBS, FRACP, PhD, Consultant Awareness of the strengths and limitations Gastroenterologist and Laboratory Head, Immunology Division, The Walter and Eliza Hall Clinical follow-up of each testing approach is vital for optimal Institute Parkville, and Gastroenterology Department, diagnosis and follow-up. The Royal Melbourne Hospital, Parkville, Vic. tyedin@ Confirming successful treatment of wehi.edu.au coeliac disease with the GFD is important Competing interests: JT-D is a co-inventor of patents Key points pertaining to the use of gluten peptides in diagnostic as the risk of complications is higher in applications and therapeutics, and non-toxic patients not achieving mucosal remission.3 Recognition and testing at-risk patients gluten for the management of coeliac disease. He • is a shareholder of Nexpep Pty Ltd and a scientific Yearly follow-up to review medical and are keys to expediting coeliac disease advisor to ImmusanT Inc, companies developing dietary progress is recommended.9 A diagnosis. novel diagnostic and therapeutic approaches for repeat gastroscopy may be considered in • Coeliac disease serology and histology coeliac disease. Provenance and peer review: Commissioned, adults after two years of starting a GFD are not accurate in people following a externally peer reviewed. to assess for mucosal healing.3 Coeliac GFD. Ask about diet when testing. disease serology is frequently used as a Positive coeliac disease serology • References surrogate marker of intestinal healing. In does not diagnose coeliac disease in 1. Hardy MY, Tye-Din JA. Coeliac disease: A adults, values correlate poorly with the isolation. The diagnosis depends on unique model for investigating broken tolerance state of the intestinal mucosa.24 A trend for showing the characteristic intestinal in autoimmunity. Clin Transl Immunology 2016;5(11):e112. normalisation is reassuring (titres generally changes and improvement on the GFD. 2. Biagi F, Corazza GR. Mortality in celiac disease. normalise on a GFD in 12 months), and • Diagnosing coeliac disease without Nat Rev Gastroenterol Hepatol 2010;7(3):158–62. persistently positive titres suggest ongoing intestinal biopsies has been considered 3. Rubio-Tapia A, Hill ID, Kelly CP, Calderwood AH, gluten exposure. In children, resolving for children, but is contentious and Murray JA, American College of Gastroenterology. ACG clinical guidelines: Diagnosis and tTG titres correlate better with mucosal requires specialist input. management of celiac disease. Am J Gastroenterol healing.25 A child who improves clinically • HLA genotyping can exclude a coeliac 2013;108(5):656–76. and normalises their serology on a GFD disease diagnosis, but has poor positive 4. Mogul D, Nakamura Y, Seo J, Blauvelt B, Bridges JF. The unknown burden and cost of celiac does not require follow-up endoscopy. predictive value. A positive result does disease in the U.S. Expert Rev Pharmacoecon Coeliac disease serology cannot detect not diagnose coeliac disease. Outcomes Res 2017;17(2):181–88.

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Biesiekierski JR, Peters SL, Newnham ED, disease. Clin Chim Acta 2010;411(13–14):931–35. gluten‑free diet can achieve the therapeutic Rosella O, Muir JG, Gibson PR. No effects goals in almost all patients with coeliac disease: of gluten in patients with self-reported A 5-year longitudinal study from diagnosis. J non-celiac gluten sensitivity after dietary Gastroenterol Hepatol 2016;31(2):342–49. reduction of fermentable, poorly absorbed, correspondence [email protected]