Secondary Logo

Journal Logo

Original Articles

Antiendomysial Antibody Test Reliability in Children With Frequent Diarrhea and Malnutrition: Is It Celiac Disease?

Gandolfi, Lenora*; Catassi, Carlo; Garcia, Steven*; Modelli, Ines Cristina*; Campos, Dioclecio Jr.*; Pratesi, Riccardo*

Author Information
Journal of Pediatric Gastroenterology and Nutrition: October 2001 - Volume 33 - Issue 4 - p 483-487
  • Free


In developed countries, the classic clinical picture of celiac disease (CD) is fairly easily recognized. In communities where the prevalence of diarrheal diseases, parasitosis, and malnutrition is low, the diagnosis of CD is one of the leading possibilities in a malnourished child with gastrointestinal symptoms. However, this picture becomes more ambiguous in developing countries. The presence of several other causes of gastrointestinal symptomatology and the still significant prevalence of chronic diarrhea and malnutrition frequently associated with parasitosis makes the diagnosis more difficult and uncertain. The multifactorial pathogenesis of malnutrition and chronic diarrhea in which nutritional, infectious, allergic, or even autoimmune factors are operative (1) generally results in a clinical picture that may be indistinguishable from that of CD. Even the jejunal mucosa lesions, which are the final common pathway through which these many noxious influences are channeled, frequently yield a similar aspect in both pathologic conditions (2,3). Presumably, serologic testing would define the correct diagnosis. In practice, this is not so simple. Diarrheal disease, malnutrition, and parasitosis are more prevalent at less than 2 years of age, and it is known that in this age group, the immunoglobulin A antiendomysial antibody (IgA EMA) test may carry up to 20% false-negative results (4). However, specificity and sensitivity of this or other serologic tests has not been determined in children with malnutrition alone or in association with chronic diarrhea and parasitosis.

Generally, the major concern regarding the validity of serologic tests, especially of the IgA EMA in malnourished children, is the possibility of false-positive results resulting from the enteropathy that is usually found in association with malnutrition, chronic intestinal infection, and parasitosis (5,6). However in this context, the appearance of false-negative results also cannot be excluded, because relative immunodeficiency may be present in malnourished children (2,7).

The primary objective of the present study was to evaluate the specificity of the IgA EMA screening test in children affected by malnutrition, frequent diarrhea, or parasitosis and secondarily to suggest criteria for the diagnosis of CD in this special group of affected children.


This project was approved by the Ethics Committee of the University of Brasilia School of Health Sciences. All children's families received written and verbal information regarding the objectives, risks, and benefits of the study. All patients in the present project were seen at the Pediatric Gastroenterology Service of the Brasilia University Hospital between October 1998 and February 2000. The larger number of children attending this hospital come from low-income families living in poor districts located in the periphery of the city. Although these children cannot be considered exceptionally disadvantaged by Brazilian standards, in their environment, the prevalence of malnutrition, parasitosis, and infectious diseases is still increased. Three hundred fifteen children were admitted to the study (183 boys, 132 girls). The age on admission varied from 6 months to 13 years and 4 months (mean age, 3 years and 5 months; standard deviation, 2.9 years; median, 2.41 years). One hundred thirty-four were less than 2 years of age and 46 were less than 1 year of age. All patients, including infants less than 1 year of age, had had at least a 3-month period of previous ingesta of gluten. Nutritional status was assessed through the classification of Waterlow et al. (8). The patients were divided into three distinct types of malnutrition: 1) acute malnutrition, in which the children showed a low weight (below the tenth percentile) for height, with the height for age equal or above the tenth percentile; 2) chronic evolutive malnutrition, in which both weight and height were low (below the tenth percentile), with the weight disproportionately low for the height; 3) chronic antecedent malnutrition, in which weight was proportional to height but height was low for age. Diarrheal episodes were classified as acute (less than 15 days of duration), persistent (more than 15 and less than 30 days of duration), and chronic (more than 30 days of duration). The clinicopathologic features of the 315 children on entry to the study are shown in Table 1.

Clinical–pathologic features of the children at entry to the study

Routine stool examination for parasites was performed in all patients by standard methods. All collected serum samples were stored at −20°C until testing. Total IgA level was determined in all sera by turbidimetric immunoquantification (COBAS MIRA; Roche Diagnostic Systems, Basel, Switzerland). An enzyme-linked immunosorbent assay technique (Bio-system, Barcelona, Spain) was used for the determination of serum immunoglobulin G antigliadin antibody levels in all children disclosing IgA values below the tenth percentile for age. An enzyme-linked immunosorbent assay method (INOVA Diagnostic Inc., San Diego, CA) was also applied to determine IgA antitransglutaminase antibody values in all children who underwent jejunal biopsy.

The presence of IgA EMA was determined on each sample by indirect immunofluorescence (9) using primate (Cebus apella) esophagus as antigenic substrate. Briefly, 4-μm cryostat sections from the distal portion of monkey esophagus were incubated at room temperature for 30 minutes with patients' sera at a 1:5 dilution. After a 10-minute washing step with phosphate-buffered saline, pH 7.2 to 7.4, the reaction was detected with fluorescein isothiocyanate rabbit antihuman IgA conjugate (Biosystems, Barcelona, Spain), applied to the sections for 30 minutes. Under the fluorescence microscope, the presence of the characteristic honeycomb-like brilliant green pattern of smooth muscle bundles was interpreted as positive (Fig. 1).

FIG. 1.
FIG. 1.:
Positive immunofluorescence showing characteristic honeycomb-like brilliant pattern of primate (Cebus apella) esophagus smooth muscle bundles. Note that the fluorescence pattern is the same as using the Rhesus monkey esophagus as an antigen substrate.

Thirty-three children in whom the severity of the clinical picture justified a prompt intervention were subjected to a peroral biopsy that was performed with a Watson pediatric capsule, samples being taken at the level of the ligament of Treitz. Biopsy results were evaluated independently by a pathologist, who was unaware of the clinical status of the patients, and by a pediatric gastroenterologist. The diagnosis of CD was based on currently accepted criteria (10). The degree of villous atrophy was graded according to the classification of Marsh (11) as normal (0), infiltrative (I), hyperplastic (II), and destructive (III). In the EMA-negative patient, the enteropathy was considered of probable allergic or environmental origin when a nonspecific infiltration of the lamina propria by mononuclear cells eventually associated with partial villous atrophy showing a patchy distribution was found at the intestinal biopsy (12).


Negative immunoglobulin G antigliadin antibody test results were obtained in four children showing low serum levels of IgA. Forty-three children showed several different parasitic infections, the most prevalent, Giardia lamblia, being present in 35 children (11.1%). The IgA EMA test results were negative in 313 children, including the 43 with parasitosis, the results being positive only in two children in whom intestinal biopsy showed typical villous flattening, crypt hypertrophy, and increased number of intraepithelial lymphocytes, confirming the diagnosis of CD (1:157). These two children also showed increased values on IgA antitransglutaminase antibody test values (61 U and 243 U, respectively; cut-off value, 20 U) and were soon after started on gluten-free diet (GFD) that was followed by a notable clinical improvement. Serologic test results reverted to normal in both these patients after 9 and 11 months of treatment, respectively.

Of the remaining 31 children who underwent intestinal biopsy because of the severity of their clinical picture, histopathologic examination disclosed a normal mucosa in 1 patient, jejunal abnormalities, considered suggestive of inflammatory or allergic enteropathy, were found in 27 (Fig. 2), and finally, grade 3 mucosal abnormalities compatible with CD were found in the remaining 3 patients. A summary of the clinical and laboratory findings of the children with biopsy results compatible with CD can be seen in Table 2.

Clinical and laboratory findings in children with CD-compatible biopsies
FIG. 2.
FIG. 2.:
Biopsy specimen from patient with negative immunoglobulin A antiendomysial antibody test results showing nonspecific inflammatory abnormalities (stain, hematoxylin–eosin; original magnification, ×400).

One of the three patients with grade 3 mucosal abnormalities, a child 1 year and 5 months old with a borderline IgA antitransglutaminase antibody value (28 U), already had a severe clinical condition complicated by septicemia and died despite institution of GFD and intensive care. The remaining two children improved steadily in response to clinical treatment and institution of GFD. One of the children, a girl, presently 2 years and 9 months old, had her GFD discontinued after 1 year. On this occasion, a control biopsy revealed only mild changes without increased intraepithelial lymphocyte count. Her IgA EMA test results continue to be normal 5 months after restoring a normal diet. The other child, currently 3 years and 3 months old, an Indian girl from a Xavante tribe living far from our center, has been reevaluated recently. Mainly because of the distance, her parents did not return to the scheduled reevaluation, 1 year after discharge. Her mother reported that the child had shown a steady improvement since the beginning of the GFD. On further questioning, she related great difficulty in maintaining a GFD mainly because of the scarcity of food options. Consequently, after few months, she had discontinued the GFD and progressively restarted a normal diet without any apparent harmful consequences. On examination, the child was clinically well, having reached during the 2-year period since her last admission normal weight and height for her age. Both repeated IgA EMA and IgA antitransglutaminase antibody test results were normal, but her control biopsy still showed some degree of villous flattening associated to enlarged crypts and increased number of intraepithelial lymphocytes.


Although false-positive results cannot be completely excluded in the two IgA EMA-positive children, this possibility seems unlikely because in both patients, a complete concordance was found between IgA EMA, IgA antitransglutaminase antibody results, and biopsy findings. In addition, in both children, a remarkable clinical improvement was observed after the institution of the GFD, and both serologic test results reverted to normal after 9 and 11 months of diet, respectively. The consistent serologic results, positive before and negative after a GFD, the biopsy finding, and satisfactory evolution on GFD compelled us to disregard the necessity of further investigations considering these children as having CD. Our results are in agreement with previous studies showing the high positive predictive value of serological CD markers in a developing country, as far as the antigliadin antibody (13) and particularly the EMA test is concerned (14).

In the present study, positive results were not detected either among children with several different degrees of mucosal lesions characteristic of environmental enteropathy or among patients with parasitosis. Even giardiasis, which has been identified as a possible cause of false-positive serologic test results (6), did not yield any abnormal results. Based on these facts, our conclusion is that chronic diarrhea and malnutrition, with or without parasitosis, do not result in the occurrence of false-positive IgA EMA test results.

However, as demonstrated by our three patients with negative IgA EMA tests and biopsy findings compatible with CD, it is quite difficult, on a first evaluation, to exclude completely the possibility of false-negative results and establish a definitive diagnosis of CD. The limited follow-up available in our three cases with a CD-like enteropathy and EMA negativity finally suggested the diagnosis of either postenteritis syndrome or transient food intolerance in these children. One hundred thirty-four of our patients were less than 2 years of age, and it is known that as many as 20% of children less than 2 years of age yield false-negative EMA results (4). In addition, it is also important to consider the possibility of immunologically compromised individuals among these malnourished children, because malnutrition is recognized as an important cause of abnormal and generally downregulated immunologic response (1,7). In these doubtful cases, human leukocyte antigens testing could be of help as a diagnostic aid. The presence of the human leukocyte antigens DQ2 or DQ8 would increase the likelihood of a positive diagnosis of CD, whereas its absence would almost certainly exclude this possibility (15).

Another aspect to be noted is that in malnourished children with histories of recurrent infections of the gastrointestinal tract, even the biopsy results can be misleading because a variable degree of mucosal abnormality is found frequently among these children. These children, after discharge return to their previous environment being subject to the same kind of stress as poor feeding and infectious and parasitic enteropathy, independent of the presence of CD. In this context, the result of a single control biopsy, showing persistence of the mucosal damage, can be misleading.

Based on our findings, we suggest a diagnostic algorithm that could help in diagnosing CD in developing countries (Fig. 3). A positive EMA test result associated with mucosal changes compatible with CD strongly suggest the diagnosis of this condition, and a long-term GFD should be started in these cases. However, EMA negativity should not be taken as evidence against this diagnosis, especially if the patient is young (less than 3 years of age) and variable damage of the small intestine mucosa is found at the biopsy. If clinically suspect, these cases could be treated initially with a GFD. If improvement does occur, the diagnosis should be definitively confirmed by a gluten challenge, as originally recommended by the European Society of Paediatric Gastroenterology, Hepatology and Nutrition (Interlaken) criteria (16). An acceptable modification would be to establish a definitive diagnosis of CD only when a previously negative IgA EMA test result reversed to positive after gluten challenge.

FIG. 3.
FIG. 3.:
Diagnostic procedure applied in the study.


1. Sullivan PB, Marsh MN, Mirakian R, et al. Chronic diarrhea and malnutrition—histology of the small intestinal lesion. J Ped Gastroenterol Nutr 1991; 12: 195–203.
2. Shiner M, Putman M, Nichols VN, et al. Pathogenesis of small-intestinal mucosal lesions in chronic diarrhea of infancy: I. A light microscopic study. J Pediatr Gastroenterol Nutr 1990; 11: 455–63.
3. Shiner M, Nichols VN, Barrish JP, et al. Pathogenesis of small-intestinal mucosal lesions in chronic diarrhea of infancy: II. An electron microscopic study. J Pediatr Gastroenterol Nutr 1990; 11: 464–80.
4. Bürgin-Wolff A, Gaze H, Hadziselimovic F, et al. Antigliadin and antiendomysium antibody determination for coeliac disease. Arch Dis Child 1991; 66: 941–7.
5. Rossi TM, Kumar V, Lerner A, et al. Relationship of endomysial antibodies to jejunal mucosal pathology: Specificity towards both symptomatic and asymptomatic celiacs. J Ped Gastroenterol Nutr 1988; 7: 858–63.
6. Chan KN, Phillips AD, Mirakian R, et al. Endomysial antibody screening in children. J Pediatr Gastroenterol Nutr 1994; 18: 316–20.
7. Smythe PM, Brereton-Stiles GG, Grace HJ, et al. Thymolymphatic deficiency and depression of cell-mediated immunity in protein-calorie malnutrition. Lancet 1971; 2: 939–43.
8. Waterlow JC. Classification and definition of protein-calorie malnutrition. Br Med J 1972; 3: 566–9.
9. Chorzelski TP, Sulej J, Tchorzewska H, et al. IgA class endomysium antibodies in dermatitis herpetiformis and coeliac disease. Ann N Y Acad Sci 1983; 420: 325–34.
10. ESPGAN Committee. Revised criteria for diagnosis of coeliac disease. Report of Working Group of European Society of Paediatric Gastroenterology and Nutrition. Arch Dis Child 1990;65:909–11.
11. Marsh M. Gluten, major histocompatibility complex, and the small intestine. Gastroenterology 1992; 102: 330–54.
12. Walker-Smith JA. Diagnostic criteria for gastrointestinal food allergy in childhood. Clin Exp Allergy 1995; 25 (suppl 1): 20–2.
13. Koshoo V, Bhan MK, Puri S, et al. Serum anti-gliadin antibody profile in childhood protracted diarrhoea due to celiac disease and other causes in a developing country. Scand J Gastroenterol 1989; 24: 1212–6.
14. Catassi C, Rätsch I-M, Gandolfi L, et al. Why is coeliac disease endemic in the people of the Sahara? Lancet 1999; 354: 647–8.
15. Partanen J. Major histocompatibility complex and coeliac disease. In: Mäki M, Collin P, Visakorpi JK, eds. Coeliac disease Tampere, Finland: Coeliac Disease Study Group, 1997; 253–64.
16. Meuwisse GW. Diagnostic criteria in coeliac disease. Acta Paeditr Scand 1970; 59: 461–3.

Celiac disease; Diarrhea; Malnutrition; Endomysial antibodies; Immunofluorescence

© 2001 Lippincott Williams & Wilkins, Inc.