Diagnostic accuracy of anti-DGP (IgG) for celiac disease

ABSTRACT

Background:

Celiac disease (CD) is an autoimmune enteropathy that is caused by the intake of gluten-containing grains in genetically susceptible humans. The gliadin and glutenin parts of wheat gluten are the essential factors that cause intestinal damage.

Objectives:

We analyzed the performance of a time-resolved immunofluorometric assay (TR-IFMA) in the diagnosis of CD in children. For this purpose, we compared the performance of IgA anti-tissue transglutaminase antibodies (IgA anti-TTG) and IgG antibodies against deamidated gliadin peptides (IgG anti-DGP) for the diagnosis of CD.

Materials and Methods:

In this cross-sectional study conducted in 2021 for a duration of about 6 months, 200 patients with suspected CD symptoms, children who needed screening due to Down syndrome or Turner syndrome, and the first-degree relatives of CD patients who underwent diagnostic evaluation were enrolled in a census study.

Results:

This study compares existing point-of-care anti-DGP Ab (IgG) and anti-TTG Ab (IgA) tests against each other using the gold standard of duodenal biopsy and pathology. Serology as a screening test was acceptable (93.6% for anti-DGP vs. 94.2% for anti-TTG) for both of them. This equivalent sensitivity of serum TTG and the DGP tests validates its potential as a basic tool for serological testing. Furthermore, endoscopy is carried out in patients positive for both.

Conclusions:

Our study showed that for the diagnosis of CD, anti-DGP antibodies had comparable characteristics with anti-TTG (IgA) in terms of diagnostic specificity and sensitivity. Specify of anti-DGP was higher than that of anti-TTG.

Introduction

Celiac disease (CD) is an autoimmune enteropathy which is caused by the intake of gluten-containing grains in genetically susceptible humans. The gliadin and glutenin parts of wheat gluten and similarly alcohol-soluble proteins in other grains (rye and barley) are the essential factors causing intestinal damage.[1] However, despite increasing knowledge of CD, global meta-analyses have shown that for one patient diagnosed, three to four patients are undetected. International cohorts have demonstrated remarkable delays in diagnosis lasting from 4 to 13 years.[2]

CD is one of the most prevalent genetic disorders that affects 1% of the world population. In susceptible individuals, gluten triggers autoimmune responses that can appear in different ways and involve multiple systems. As these diverse patterns can impose diagnostic challenges, it is crucial that clinicians keep CD in their mind when evaluate their patients. The cultivation of wheat was first done in the “fertile crescent” zone extending from modern Turkey to Iran. The literature has mentioned CD in this area and reports of high prevalence of CD in populations of Jordan, Turkey, Lebanon, Iran, Tunisia, Egypt, Israel, and Kuwait.[3] The disease was not considered to be prevalent in Iran up to a decade ago; but using serological tests for diagnosis of CD, several articles have been published on the prevalence of CD in Iran. This prevalence is different among subgroups and varies from 0.5% among schizophrenia patients to 12% among those with irritable bowel syndrome (IBS).[4] The differential diagnosis of CD consists of cystic fibrosis, autoimmune enteropathy, pediatric IBS, inflammatory bowel disease (Crohn disease), intolerance of protein, eosinophilia gastroenteritis, parasites such as giardiasis, and bacterial and viral gastroenteritis.[5] Antibodies against an individual’s own proteins (autoantibodies) are indicators of autoimmunity; There are several tests to diagnose CD and the most commonly used is anti-tissue transglutaminase antibodies (anti-TTG).[6] It has been shown that the sensitivity of anti-TTG antibody is indistinctive in patients with mild intestinal damage, which is common in patients with a low gluten diet intake and in family members of the patients. Another group that may have false-negative results by using autoantibodies are children younger than 3 years, who have low autoimmune responses. In this population of patients, the combination of anti-TTG and IgG antibodies against deamidated gliadin peptides (anti-DGP) may modify the detection of CD. The diagnostic value of anti-DGP as a first-line diagnostic test has not been studied. Its usage has been common in young people and in those with IgA total deficiency, and whether it is valuable as the first diagnostic test in old age or not is debatable.[7] The gold standard for CD diagnosis is represented by a combination of mucosal changes detected by duodenal biopsy and by positivity of serological tests (anti-TTG antibodies, anti-endomysium antibodies [anti-EMA], and deamidated gliadin peptide [DGP] antibodies). Despite the progress that has been made in serologies, no antibody test is now available with 100% sensitivity and specificity; therefore, intestinal biopsy is needed for providing a definite diagnosis.[8] Increased intraepithelial lymphocytes (IELs) are the basic and most sensitive marker of the influence of gluten on the small bowel mucosa; therefore, it is the main histological finding of CD. Increase in here means >40 lymphocytes per 100 upper crypt or surface enterocytes. The majority of them are CD2+, CD3+, and CD8+ T cells. In latent forms of CD, usually increase in IELs is the only abnormal feature and villous architectures and lamina propria cells are in the normal range. Counting the lymphocytes should be performed on villi with good orientation in cuts with 3–4 μm thickness.[9]

CD severity diagnosis on duodenal histopathologic images is defined by Marsh classification which includes (1) infiltrative lesions (IELs more than 25/100 epithelial cells) (Marsh 1), (2) crypt hyperplasia (Marsh 2), and (3) villous atrophy with variable severity (Marsh 3 a, b, c).[10]

During the coronavirus disease 2019 (COVID-19) pandemic, an impermanent decrement of the TTG–IgA threshold for biopsy-sparing approach seems conceivable, avoiding missed diagnosis and complications. In the near future, a critical revision of the European society for paediatric gastroenterology hepatology and nutrition (ESPGHAN) biopsy-free criteria for CD diagnosis is needed.[11] Also, having stronger findings for doing endoscopy in patients is logical during the covid period. The prevalence of CD in Iran, the importance of the disease, and the great financial and physical burden that this disease has on children are the main reasons for conducting further studies. The importance of timely diagnosis and treatment of the disease is emphasized. Also, considering that the initial and important diagnostic step after clinical suspicion is serological tests, finding an accurate test with high positive predictive value and low error probability is very valuable. This problem became even more important during the COVID-19 period because endoscopy was associated with the risk of covid transmission and limitations which were defined for doing endoscopy had a negative effect on the diagnosis of CD, thus powerful serological tests can prevent missing the celiac disese.

Materials and Methods

This is an analytical cross-sectional study of patients who attended the Division of Pediatric Gastroenterology of Akbar Hospital for an esophagogastroduodenoscopy (EGD) due to suspicion of CD. To see if the test is valuable, we compare it with the gold standard, which is a biopsy, to see if it can predict histological changes in different patients [Table 1].

Table 1.

Endoscopic findings in patients

	Frequency	Percentage	Cumulative percentage
Endoscopy
1. Normal	93	46.5	46.5
2. Nodularity	6	3	49.5
3. Scalloping	95	47.5	97
4. Atrophy	6	3	100
Total	200	100

In a cross-sectional study conducted in 2021 for a duration of 6 months, 200 patients with suspected CD symptoms, such as impaired growth, abnormal stools, abdominal distension, pallor, muscle wasting, poor appetite, and abnormal behavior, and extraintestinal symptoms, such as unexplained anemia, insulin-dependent diabetes mellitus, short stature, autoimmune endocrinopathies, aphthous stomatitis, IgA deficiency, enamel hypoplasia, connective tissue disorders, infertility, intractable seizures, ataxia, polyneuropathy, hypertransaminasemia, osteoporosis, and alopecia, and children who needed screening due to Down syndrome or Turner syndrome and their first-degree relatives who underwent diagnostic evaluation were enrolled in the study [Table 2].

Table 2.

Chief complaints and reasons for suspecting celiac disease and doing endoscopy and biopsy

	Frequency	Percentage	Cumulative percentage
Valid
Growth disorder	112	56.0	56.0
Abdominal pain	34	17.0	73.0
Positive relatives	8	4.0	77.0
Skin	1	0.5	77.5
Maturity delay	11	5.5	83.0
Diarrhea	8	4.0	87.0
Anemia	4	2.0	89.0
Diabetes	20	10.0	99.0
Hypothyroidism	2	1.0	100.0
Total	200	100	100

In all children, the levels of total IgA and IgA anti-DGP were measured by using enzyme-linked immunosorbent assay (ELISA) (QUANTA Lite Celiac), with a cut-off value >15 IU, and IgA anti-TTG was measured by using the ELISA (QUANTA), with a cut-off value >15 U/L, and positive cases were candidates for doing endoscopy and taking the biopsy. Anti-DGP IgG is an ELISA-based, automated, in vitro test system for the quantitative determination of IgG antibodies against deamidated gliadin protein (DGP) epitopes in human serum or plasma. These assays are used to support a diagnosis of CD. Tests and blood sampling were done in Sadra laboratory; the tests were done on the routine blood samples of the vein.

For all patients who had an EGD, six biopsies were done (four in the second part of the duodenum and two in the duodenal bulb). Histopathologic results were described using the Marsh–Oberhuber classification: Marsh 0: normal mucus membrane; Marsh 1: increased IELs; Marsh 2: increased IELs with crypt hyperplasia; and Marsh 3: atrophic villi. Patients were considered to have CD if they were classified as Marsh 1, 2, and 3. If the biopsy evidence was consistent with the celiac histological diagnosis including increased number of IELs (more than 25 per 100 enterocytes), elongation of the crypts, and partial to total villous atrophy, the diagnosis was made and anti-DGP samples were evaluated for specificity, sensitivity, positive predictive value, and negative predictive value.[12] The sample should be handled gently with gloved hands or using smooth or rubber-tipped forceps. Foam pad inserts for specimen cassettes, dental wax, or lens paper can be used to orient the specimen before placing it in a fixative. Exclusion criteria for this study were patient dissatisfaction, dissatisfaction with endoscopy after positive test, and failure to perform anti-DGP. Inclusion criteria were all suspected celiac patients including children with gastrointestinal symptoms and extraintestinal symptoms. In this study, we measured the anti-TTG (IgA) and anti-DGP (IgG) values in all suspected patients. Endoscopy was performed for all patients who tested positive, and the results were compared for calculating positive and negative predictive values.

The following definitions and computations of Bayesian diagnostic parameters were used to compare the different CD populations in the microarray based on standard 2 × 2 concordance table analysis with A = true positives, B = false positives, C = false negatives, and D = true negatives:

• –TPR is the true positive rate or sensitivity = A/(A + C)

• –TNR is the true negative rate or specificity = D/(B + D)

• –PPV is the positive predictive value or precision = A/(A + B)

• –Accuracy (overall agreement) is the proportion correctly classified = (A + D)/(A + B + C + D)

Sample size: Based on previous studies and prevalence of CD (up to 23%) as reported in a journal of pediatrics gastroenterology and nutrition (JPGN) article (1), considering the significance level of 0.95 and an acceptable error of 0.05, the sample size was calculated using the formula and found to be 196 people.

This research R.MUMS.MEDICAL.REC.1400.226 code was approved in 2021 in the Mashhad Medical School.

Results

The mean (standard deviation) of age was 8.44 (3.68) and age range was 1–17 years. Gender distribution among our patients showed 73 boys and 127 girls and the percentages were 36.5 and 63.5, respectively.

For anti-DGP (IgG), the results showed the following: sensitivity: 93.6%, specificity: 65.5%, positive predictive value: 94.1%, and negative predictive value: 63.3%. For anti-DGP (IgA), the values were as follows: sensitivity: 94.2%, specificity: 6.9%, positive predictive value: 85.6%, negative predictive value: 16.7%. Given the area below the receiver operating characteristics (ROC) curve (0.795) and the reported p value (P < 0.001), the diagnostic accuracy of the anti-DGP test is acceptable [Figure 1].

Figure 1.

ROC curve, the diagnostic accuracy of the anti-DGP test

Discussion

This is a study that compares existing point-of-care for anti-DGP Ab (IgG) and anti-TTG Ab (IgA) tests against each other using the gold standard of duodenal biopsy and pathology. Serology as a screening test was acceptable for both of them (93.6% anti-DGP vs. 94.2% for anti-TTG). This equivalent sensitivity of serum anti-TTG and anti-DGP test validates their potential as a basic tool for serolo gical testing. Furthermore, endoscopy is carried out in patients positive in both tests. Using this point-of-care test in endoscopy before all procedures has the potential to significantly reduce the number of missed cases at endoscopy. The prevalence of CD in Iran is different in subgroups and varies from 0.5% among patients with schizophrenia to 12% in IBS patients.[4] Missed diagnosis of CD may lead to many side effects in patients. First, it may result in patients experiencing persistent symptoms that could have been treated with a gluten-free diet, thus avoiding unnecessary investigations and clinic visits with associated costs, and second, it may result in a delay in beginning an effective gluten-free diet, which can aggravate the risk of CD complications such as osteoporosis, anemia, and even lymphoma.[2]

In line with our study, Ortiz et al.[6] reported in their study that IgG anti-DGP antibodies exhibited a higher sensitivity and specificity than IgA antibodies. Also, as detailed by Villalta et al.,[13] IgG anti-DGP antibodies had a better performance than IgA antibodies, especially in patients with IgA deficiency and younger than 2 years.

Kurppa et al.[14] showed that the sensitivity of anti-DGP was more than anti-TTG Ab even in celiac patients in early-stage CD with normal villous morphology. On the basis of these results, anti-DGP would seem to offer an acceptable new method for case finding and follow-up.

Velikova et al.[15] claimed that anti-DGP antibodies have comparable and even more acceptable characteristics in terms of diagnostic specificity and sensitivity for the identification of patients with CD, especially those with IgA deficiency, as well as identifying other diseases associated with sensitivity to gluten, as they help in moni toring the effect of the gluten-free diet. The combination of autoantibodies related to CD, including the anti-DGP antibodies, improves the diagnosis and follow-up of these patients.

Abdulrahim et al.[16] reported that anti-DGP–IgG assay did not add to improve the performance of anti-TTG–IgA. Their results were different from ours.

Aaron et al.[17] mentioned that it seems that the trend that recommended anti-DGP usage for CD screening and diagnosis, during the first decade of the current millennium (2000–2012) changed its direction. They also reported that based on multiple studies and reviews, it is found that the anti-DGP antibody is not as useful as a nti-TTG as a prime serological marker. Their results were not consonant with ours.

Utilization

The importance of timely diagnosis and treatment of the disease is emphasized. Also, considering that the initial and important diagnostic step after clinical suspicion is serological tests, finding an accurate test with high positive predictive value and low error probability is very valuable.

Good and acceptable sample size and appropriate confirmation of cases with pathology are the strengths of the study. Lack of a control group for more comparisons is one of the weaknesses of the study.

Suggestions

We recommend to conduct further studies using case and control groups to compare the simultaneous use of tests and its effect on improving patient identification. We also suggest to categorize groups by age to determine the best test for each one. Also, using other serological tests and comparing them allows us to take the best decision about more aggressive measures.

Rapid tests can also be used in future studies to expedite diagnostic and therapeutic works.

Also, reviewing tests for therapeutic follow-up and comparing the severity of clinical and endoscopic symptoms and pathology with the serum levels of the tests is one of the things that can be done with anti-DGP (IgG) use. As mentioned before, the sensitivity of the anti-TTG antibody reduces in patients with mild intestinal damage, which is frequent in individuals with a low gluten intake and in family members of celiac patients. Another population that may present false-negative results when using autoantibodies includes children younger than 3 years and who have a low autoimmune response. In this group of patients, the combination of anti-TTG antibodies and IgG antibodies against DGPs (IgG anti-DGP) may improve the detection of CD. The diagnostic value of anti-DGP as a primary diagnostic test has not been studied much and its use has been mostly in young people and in those with IgA total deficiency. Whether it is valuable as the first diagnostic test in old age or not is debatable. Our study showed the value of this serology in those who are susceptible to CD.

Conclusion

In conclusion, our study showed that for the diagnosis of CD, anti-DGP antibodies have shown comparable characteristics in terms of diagnostic specificity and sensitivity with anti-TTG (IgA). Due to the prevalence of CD in Iran, the importance of this disease and the great financial and physical burden that this disease has on children, the value of timely diagnosis and treatment of the disease is emphasized here. Also, considering that the initial and important diagnostic step after clinical suspicion is serological tests, finding an accurate test with high positive predictive value and low error probability is very valuable.

We can use anti-DGP besides instead of anti-TTG (IgA) for detecting CD, especially in children younger than 3 years who have a low autoimmune response.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.