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SERONEGATIVE CELIAC DISEASE IN BRAZILIAN PATIENTS: A SERIES OF CASES

Doença celíaca soronegativa em pacientes brasileiros: uma série de casos

ABSTRACT

BACKGROUND:

Celiac disease (CD) is an autoimmune disease characterized by immune reaction mostly to wheat gluten. The diagnosis is based on clinical, serological and histological findings in patients ingesting gluten. Cases that the clinical profile indicates CD and the autoantibodies are negative bring so a dilemma for the professional, as the risk of missed the diagnosis or a delay at the same.

OBJECTIVE:

To show the importance of correct diagnosis of cases with seronegative celiac disease (SNCD).

METHODS:

Ten cases of SNCD Brazilian patients were retrospectively studied (2013 to 2019). Data of clinical complaints, autoantibodies, IgA serum levels, histological findings and HLA-DQ2/DQ-8 were compiled. Dual-X densitometry, delay at diagnosis, previous autoimmune diseases and family history of CD were also checked.

RESULTS:

All SNCD patients presented clinical symptoms of CD, with confirmed diagnosis by histological findings of the duodenal mucosa and HLA-DQ2 and/or HLA-DQ8 positivity. All patients had normal IgA levels and negative autoantibodies (IgA-anti-transglutaminase and anti-endomysial). Dual-X densitometry detected osteopenia in two women and osteoporosis in two males, all with low levels of vitamin D. Delay diagnostic ranged from 1 to 19 years. Familiar occurrence of CD was reported in 40% of the cases. After one year of gluten-free diet, eight patients refer improve of symptoms, while duodenal biopsies, done in five cases, showed histological improvement.

CONCLUSION:

Patients who demonstrate the clinical profile of celiac disease with negative serology and normal levels of IgA, especially those who have family members with celiac disease, should be submitted to duodenal biopsies to look for histological findings.

Keywords:
Celiac disease; autoantibodies; diagnosis

RESUMO

CONTEXTO:

A doença celíaca (DC) é uma doença autoimune caracterizada por reação imune principalmente ao glúten do trigo. O diagnóstico é baseado em achados clínicos, sorológicos e histológicos em pacientes que ingerem glúten. Casos em que o perfil clínico indica DC e os autoanticorpos são negativos trazem um dilema para o profissional, como o risco de não realizar ou atrasar o diagnóstico da DC.

OBJETIVO:

Mostrar a importância do diagnóstico correto de casos com doença celíaca soronegativa (DCSN).

MÉTODOS:

Dez casos de pacientes brasileiros com DCSN foram estudados retrospectivamente (2013 a 2019). Foram compilados dados de queixas clínicas, autoanticorpos, níveis séricos de IgA, achados histológicos e HLA-DQ2 / DQ-8. Densitometria, atraso no diagnóstico, doenças autoimunes prévias e histórico familiar de DC também foram verificados.

RESULTADOS:

Todos os pacientes com DCSN apresentaram sintomas clínicos de DC, com diagnóstico confirmado por achados histológicos da mucosa duodenal e positividade para HLA-DQ2 e/ou HLA-DQ8. Todos os pacientes apresentavam níveis normais de IgA e autoanticorpos negativos (IgA-anti-transglutaminase e anti-endomisial). A densitometria detectou osteopenia em duas mulheres e osteoporose em dois homens, todos com baixos níveis de vitamina D. O atraso no diagnóstico variou de 1 a 19 anos. A ocorrência familiar de DC foi relatada em 40% dos casos. Após 1 ano de dieta isenta em glúten, oito pacientes referem melhora dos sintomas, enquanto as biópsias duodenais, realizadas em cinco casos, mostraram melhora histológica.

CONCLUSÃO:

Pacientes que apresentam quadro clínico de doença celíaca com sorologia negativa e níveis normais de IgA, principalmente aqueles que possuem familiares com doença celíaca, devem ser submetidos à biópsia duodenal para pesquisa de achados histológicos.

Palavras-chave:
Doença celíaca; auto anticorpos; diagnóstico

INTRODUCTION

Celiac disease (CD) is a chronic immune-mediated disease triggered by the exposure to dietary gluten with strongly genetical influence, specially related to the presence of HLA DQ2/DQ8.11. Caio G, Volta U, Sapone A, Leffler DA, De Giorgio R, Catassi C, Fasano A. Celiac disease: a comprehensive current review. BMC Med. 2019;17:142. Usually the diagnosis is based on clinical symptoms, positivity of autoantibodies and histologic findings in patients ingesting gluten11. Caio G, Volta U, Sapone A, Leffler DA, De Giorgio R, Catassi C, Fasano A. Celiac disease: a comprehensive current review. BMC Med. 2019;17:142.,22. Rubio-Tapia A, Hill ID, Kelly CP, Calderwood AH, Murray JA. American College of Gastroenterology clinical guideline: diagnosis and management of celiac disease. Am J Gastroenterol. 2013;108:656-77.. However, there are cases when the clinical profile indicates CD and the autoantibodies are negative, causing a dilemma for the professional33. DeGaetani M, Tennyson CA, Lebwohl B, Lewis SK, Abu Daya H, Arguelles-Grande C, Bhagat G, Green PH. Villous atrophy and negative celiac serology: A diagnostic and therapeutic dilemma. Am J Gastroentrol. 2013;108:647-53.. CD diagnosis can be missed if the serology is negative44. Farina MH, Kumar Mandhwani R, Hassan Luck N, Abbas Z, Mubarak M, Laeeq SM, Tasneem AA. Clinicopathological Study of Seronegative Celiac Disease in Adults in Pakistan: A Pilot Study. Middle East J Dig Dis. 2017;9:94-9.. The objective of this study is to present a series of cases with seronegative celiac disease (SNCD).

METHODS

This study was approved by the Ethics Committee of the Evangelical Beneficent Society of Curitiba (CAAE 84793318.0.0000.0103). This is a retrospective study performed through a survey of clinical charts. The same physician attended all the patients in a single private practice in the city of Curitiba, Brazil, during the period 2013 to 2019.

All included patients were diagnosed with CD based on clinical complaints, HLA-DQ2 and/or HLADQ-8 positivity and confirmation by histological findings of the duodenal mucosa, according to the Marsh classification55. Marsh MN. Gluten, major histocompatibility complex, and the small intestine. A molecular and immunobiologic approach to the spectrum of gluten sensitivity (“celiac sprue”). Gastroenterology. 1992;102:330-54.. The complaints registered were aphtha, gastroesophageal reflux, epigastric pain, indigestion, nausea, vomiting, flatulence, abdominal pain, diarrhea, constipation and abdominal distension, depression and anxiety. In addition, weigh­ting, routine laboratory tests and Dual-X ray absormetry was done. Delay at diagnosis, family history of CD and previous autoimmune diseases was checked. Differential diagnosis with parasitic, infectious or inflammatory diseases, and the use of drugs, were ruled out as preconized by anamnesis and appropriate tests.

All the patients were simultaneously tested for serum levels of IgA and for autoantibodies class IgA-anti-tissue transglutaminase and IgA anti-endomysial. Upper endoscopy was performed with two or three biopsies of the duodenal bulb and at least four specimens post-bulbar22. Rubio-Tapia A, Hill ID, Kelly CP, Calderwood AH, Murray JA. American College of Gastroenterology clinical guideline: diagnosis and management of celiac disease. Am J Gastroenterol. 2013;108:656-77.. Standard haematoxylin and eosin (H&E) was used for the analysis of the fragments. Experienced gastrointestinal pathologist referred results according to Marsh classification55. Marsh MN. Gluten, major histocompatibility complex, and the small intestine. A molecular and immunobiologic approach to the spectrum of gluten sensitivity (“celiac sprue”). Gastroenterology. 1992;102:330-54.. HLA-DQ2 and HLA-DQ8 presence were tested using DNA amplified by polymerase chain reaction.

After diagnosis, the patients were informed about CD and how to adhere to a gluten-free diet (GFD). After one year, they were clinically and laboratory reevaluated and a second duodenal biopsies were performed.

RESULTS

Were studied ten Caucasian Brazilian patients, seven women and three men, median age 30.5 years (range 16 to 68 years), who presented clinical symptoms suggestive of CD and negative autoantibodies (IgA-antiendomysial and IgA-anti tissue transglutaminase). Familiar occurrence of CD was reported in 40% of the cases (three mothers and one sister). The demographic, clinical and laboratorial findings were demonstrated in Table 1.

TABLE 1
Demographic, clinical and laboratorial findings of patients with celiac disease seronegative.

In relation to symptoms, abdominal pain, abdominal distension, flatulence and gastroesophageal reflux were referred in 80%; depression in 60%; epigastric pain in 50%; diarrhea, constipation, aphtha and indigestion in 30%; nausea and vomit in 20%.

In nine cases a Dual-X densitometry was performed. Five patients had normal results. Osteopenia was observed in two cases (both women, with 18 and 57 years), while osteoporosis was detected in two males (16 and 68 years). All the patients had low levels of vitamin D.

Table 1 also shows the HLA typing, delay diagnostic, endoscopic and histological findings at diagnosis and after GFD. After one year of GFD, eight patients were reevaluated. Adherence was report by all, referring improvement of symptoms, with return of them only when gluten was ingested inadvertently. All the laboratory tests were normal. Duodenal biopsies were done in five cases, showing histological improvement in four cases.

DISCUSSION

This study presented a series of cases of SNCD, demonstrating that, although not frequent, can occur in the clinical practice. Clinical manifestations of CD have a wide range of symptoms and some patients can show oligo symptomatic form, and the serological tests are useful for identifying CD. The SNCD is characterized by the lack of demonstrable serological markers along with clinical signs of malabsorption and atrophy of the mucosa in patients ingesting gluten66. Volta U, Caio G, Boschetti E, Giancola F, Rhoden KJ, Ruggeri E, Paterini P, De Giorgio R. Seronegative celiac disease: Shedding light on an obscure clinical entity. Dig Liver Dis. 2016;48:1018-22.. Even with a high sensitivity of tests, a minority of patients with CD are seronegative, with prevalence of 1.03% among all CD patients to 28% in latent CD77. Giorgio F, Principi M, Losurdo G, Piscitelli D, Iannone A, Barone M, Amoruso A, Lerardi E, Di Leo A. Seronegative celiac disease and immunoglobulin deficiency: Where to look in the submerged iceberg? Nutrients. 2015;7:7486-504..

All diagnostic serologic testing should be done with the patients on a gluten-containing diet22. Rubio-Tapia A, Hill ID, Kelly CP, Calderwood AH, Murray JA. American College of Gastroenterology clinical guideline: diagnosis and management of celiac disease. Am J Gastroenterol. 2013;108:656-77.. It is recommended that cases of patients ingesting gluten with negative serology, but with positive histology for CD and compatible HLA, may be treated as having CD88. Huzby S, Murray JA, Katzka DA. AGA Clinical Practice Update on Diagnosis and Monitoring of Celiac Disease: Changing Utility of Serology and Histologic Measures: Expert Review. Gastroenterology. 2019;156:885-9..

In CD, the production of autoantibodies occurs in the intestinal mucosa, as evidenced by the presence of immune complexes by immunofluorescence test77. Giorgio F, Principi M, Losurdo G, Piscitelli D, Iannone A, Barone M, Amoruso A, Lerardi E, Di Leo A. Seronegative celiac disease and immunoglobulin deficiency: Where to look in the submerged iceberg? Nutrients. 2015;7:7486-504.. These autoantibodies usually cross the mucosa and reach the blood vessels. However, in SNCD these antibodies may stay in the lamina propria rather than passing into the bloodstream77. Giorgio F, Principi M, Losurdo G, Piscitelli D, Iannone A, Barone M, Amoruso A, Lerardi E, Di Leo A. Seronegative celiac disease and immunoglobulin deficiency: Where to look in the submerged iceberg? Nutrients. 2015;7:7486-504.. Immaturity of plasma cells is another hypothesis in the mechanism of SNCD99. Lerardi E, Losurdo G, Piscitelli D, Giorgio F, Sorrentino C, Principi M, Montenegro L, Amoruso A, Di Leo A. Seronegative celiac disease: where is the specific setting? Gastroenterol Hepatol Bed Bench. 2015;8:110-6..

In this study, we found in all cases HLA compatible with CD, supporting the diagnosis of CD1010. Brown NK, Guandalini S, Semrad C, Kupfer SS. A Clinician’s Guide to Celiac Disease HLA Genetics. Am J Gastroenterol . 2019;114:1587-92.. CD occurred in subjects who presented HLA-DQ2 and/or DQ8 gene loci, at any age, following ingestion of gluten-containing food1010. Brown NK, Guandalini S, Semrad C, Kupfer SS. A Clinician’s Guide to Celiac Disease HLA Genetics. Am J Gastroenterol . 2019;114:1587-92.. DQ2 is detected in 95% and DQ8 in 5% of the patients with CD, and although these haplotypes are very common in the general population, with a mean prevalence of 20%, although only a minority develop CD, presenting a high negative predictive value1010. Brown NK, Guandalini S, Semrad C, Kupfer SS. A Clinician’s Guide to Celiac Disease HLA Genetics. Am J Gastroenterol . 2019;114:1587-92.. Thus, in SNCD, HLA testing is useful to support diagnosis when biopsy or serology are equivocal.

The symptoms referred by the patients were similar to the reported in Brazilian patients with seropositive CD1111. Lima RF, Maria da Silva Kotze L, Kotze LR, Chrisostomo KR, Nisihara R. Gender-Related Differences in Celiac Patients at Diagnosis. Arch Med Res. 2019;50:437-41.. Nutritional profile of adults with CD is important, however, in our patients 60% presented normal weight, 10% were above and 30% present overweight. Awareness of obesity needs to be considered to avoid fail or delayed in the diagnosis of CD1212. Dickey W, Kearney N. Overweight in celiac disease: prevalence, clinical characteristics, and effect of a gluten-free diet. Am J Gastroenterol . 2006;101:2356-9..

Anemia due to iron and vitamin B12 deficiency was verified in 40% of our patients and low levels of vitamin D in all, with bone disease in four, inclusive two young patients. Deficiency of micronutrients could require dietary supplementation in the beginning of treatment until a strict GFD proved improvement1313. Rondanelli M, Faliva MA, Gasparri C, Peroni G, Naso M, Picciotto G, et al. Micronutrients Dietary Supplementation Advices for Celiac Patients on Long-Term Gluten-Free Diet with Good Compliance: A Review. Medicina (Kaunas). 2019;3:55-337..

In Brazilians patients with seropositive CD, Silva et al. demonstrated that 69% at diagnosis had low bone mineral density, being 36 (35.6%) younger than 30 years1414. Silva JT, Nisihara RM, Kotze LR, Olandoski M, Kotze LM. Low bone mineral density in Brazilian patients at diagnosis of celiac disease. Arq Gastroenterol. 2015;52:176-9., values similar to observed in the present investigation, which low bone mineral density was detected in four cases.

Although all of our patients had symptoms of CD, the observed diagnostic delay was long in almost all cases. The fact that serological tests are negative can influence this fact, given that such tests are used as screening. In patients with SNCD, the difficulty in diagnosing CD is certainly greater and further investigation is necessary.

There was not strict correlation between endoscopy and histologic findings since duodenal mucosa can show patchy lesions1515. Kotze LMS, Kotze LR, Moreno I, Nisihara R. Immune mediated diseases in patients with celiac disease and their relatives: A comparative study of age and sex. Arq Gastroenterol . 2018;55:346-51.. Our findings are in accordance with this proposition.

Clinical and laboratory parameters improve in patients with SNCD after a strict adherence to a GFD, similarly to patients with seropositive CD, as was find in our study. Especially important is the improvement of intestinal mucosa observed in the most of patients in the duodenal specimens obtained after GFD as demonstrated in the present study. Histological changes after a GFD in patients with and without villous atrophy strongly support diagnosis of SNCD22. Rubio-Tapia A, Hill ID, Kelly CP, Calderwood AH, Murray JA. American College of Gastroenterology clinical guideline: diagnosis and management of celiac disease. Am J Gastroenterol. 2013;108:656-77..

Immune mediated disease (IMD) are reported as comorbidities in patients with CD before, at diagnosis or in the follow-up1515. Kotze LMS, Kotze LR, Moreno I, Nisihara R. Immune mediated diseases in patients with celiac disease and their relatives: A comparative study of age and sex. Arq Gastroenterol . 2018;55:346-51.. In the present study, we confirm this fact. Before the diagnosis of CD, one man reported hypothyroidism after Hashimoto thyroiditis, scleroderma in plaques and alopecia areata. After a GFD, three patients presented immune mediated disorders in a one-year reevaluation (two endometriosis, one hyperthyroidism and one lymphocytic colitis). If diagnosis of CD and subsequent dietary treatment can prevent autoimmune diseases remains controversial1616. Lundin KE, Wijmenga C. Coeliac disease and autoimmune disease-genetic overlap and screening. Nat Rev Gastroenterol Hepatol. 2015;12:507-15..

This study had some limitations. Three patients did not perform the second biopsy. It is need to considerer that in clinical practice is difficult that patients with an excellent response to a gluten-free diet agree to be submitted to a second upper endoscopy.

Concluding, patients presenting clinical profile of CD with negative serology and normal levels of IgA, especially those with family members with CD, should be submitted to duodenal biopsies to look for histological findings.

REFERENCES

  • 1
    Caio G, Volta U, Sapone A, Leffler DA, De Giorgio R, Catassi C, Fasano A. Celiac disease: a comprehensive current review. BMC Med. 2019;17:142.
  • 2
    Rubio-Tapia A, Hill ID, Kelly CP, Calderwood AH, Murray JA. American College of Gastroenterology clinical guideline: diagnosis and management of celiac disease. Am J Gastroenterol. 2013;108:656-77.
  • 3
    DeGaetani M, Tennyson CA, Lebwohl B, Lewis SK, Abu Daya H, Arguelles-Grande C, Bhagat G, Green PH. Villous atrophy and negative celiac serology: A diagnostic and therapeutic dilemma. Am J Gastroentrol. 2013;108:647-53.
  • 4
    Farina MH, Kumar Mandhwani R, Hassan Luck N, Abbas Z, Mubarak M, Laeeq SM, Tasneem AA. Clinicopathological Study of Seronegative Celiac Disease in Adults in Pakistan: A Pilot Study. Middle East J Dig Dis. 2017;9:94-9.
  • 5
    Marsh MN. Gluten, major histocompatibility complex, and the small intestine. A molecular and immunobiologic approach to the spectrum of gluten sensitivity (“celiac sprue”). Gastroenterology. 1992;102:330-54.
  • 6
    Volta U, Caio G, Boschetti E, Giancola F, Rhoden KJ, Ruggeri E, Paterini P, De Giorgio R. Seronegative celiac disease: Shedding light on an obscure clinical entity. Dig Liver Dis. 2016;48:1018-22.
  • 7
    Giorgio F, Principi M, Losurdo G, Piscitelli D, Iannone A, Barone M, Amoruso A, Lerardi E, Di Leo A. Seronegative celiac disease and immunoglobulin deficiency: Where to look in the submerged iceberg? Nutrients. 2015;7:7486-504.
  • 8
    Huzby S, Murray JA, Katzka DA. AGA Clinical Practice Update on Diagnosis and Monitoring of Celiac Disease: Changing Utility of Serology and Histologic Measures: Expert Review. Gastroenterology. 2019;156:885-9.
  • 9
    Lerardi E, Losurdo G, Piscitelli D, Giorgio F, Sorrentino C, Principi M, Montenegro L, Amoruso A, Di Leo A. Seronegative celiac disease: where is the specific setting? Gastroenterol Hepatol Bed Bench. 2015;8:110-6.
  • 10
    Brown NK, Guandalini S, Semrad C, Kupfer SS. A Clinician’s Guide to Celiac Disease HLA Genetics. Am J Gastroenterol . 2019;114:1587-92.
  • 11
    Lima RF, Maria da Silva Kotze L, Kotze LR, Chrisostomo KR, Nisihara R. Gender-Related Differences in Celiac Patients at Diagnosis. Arch Med Res. 2019;50:437-41.
  • 12
    Dickey W, Kearney N. Overweight in celiac disease: prevalence, clinical characteristics, and effect of a gluten-free diet. Am J Gastroenterol . 2006;101:2356-9.
  • 13
    Rondanelli M, Faliva MA, Gasparri C, Peroni G, Naso M, Picciotto G, et al. Micronutrients Dietary Supplementation Advices for Celiac Patients on Long-Term Gluten-Free Diet with Good Compliance: A Review. Medicina (Kaunas). 2019;3:55-337.
  • 14
    Silva JT, Nisihara RM, Kotze LR, Olandoski M, Kotze LM. Low bone mineral density in Brazilian patients at diagnosis of celiac disease. Arq Gastroenterol. 2015;52:176-9.
  • 15
    Kotze LMS, Kotze LR, Moreno I, Nisihara R. Immune mediated diseases in patients with celiac disease and their relatives: A comparative study of age and sex. Arq Gastroenterol . 2018;55:346-51.
  • 16
    Lundin KE, Wijmenga C. Coeliac disease and autoimmune disease-genetic overlap and screening. Nat Rev Gastroenterol Hepatol. 2015;12:507-15.
  • Disclosure of funding: no funding received

Publication Dates

  • Publication in this collection
    05 July 2021
  • Date of issue
    Apr-Jun 2021

History

  • Received
    23 June 2020
  • Accepted
    06 Jan 2021
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