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Celiac Disease Screening among Healthy Blood Donors in Hungary Zöliakie-Screening bei gesunden Blutspendern in Ungarn Authors
M. Sárdy1, 2, Z. Kornseé1, D. Kelemen1, S. Papp1, M. Medvecz1, S. Kárpáti1
Affiliations
1
Department of Dermatology, Venereology and Dermatooncology, Semmelweis University, Budapest Department of Dermatology and Allergology, Ludwig Maximilian University, Munich, Germany
Schlüsselwörter
Zusammenfassung
Abstract
"
!
!
Hintergrund: Die Zöliakie ist eine häufige, chronische systemische Autoimmunerkrankung in Europa. Ihre Prävalenz bei Kindern in der Allgemeinbevölkerung Ungarns liegt bei 1,2 – 1,4 %. Bislang gibt es keine Daten hinsichtlich der Prävalenz bei Erwachsenen in Ungarn. Ziele: Analyse der Seren von Erwachsenen in Ungarn zur Ermittlung der Prävalenz der Zöliakie. Methoden: Plasmaproben von 4155 gesund erscheinenden Blutspendern wurden anonymisiert und hinsichtlich zirkulierender IgA-Autoantikörper gegen Gewebstransglutaminase mittels eines hoch sensitiven ELISA untersucht. Die positiven Ergebnisse wurden mithilfe eines EndomysiumAntikörpertests verifiziert. Ergebnisse: Der Endomysium-Antikörpertest war bei 25 Proben positiv, insofern scheint die Prävalenz der Zöliakie bei mindestens etwa 0,6 % (1:166) zu liegen. Da die Proben anonymisiert getestet wurden, konnten die Betroffenen nicht weiter untersucht werden. Schlussfolgerungen: Die erste serologische Screeningstudie bei gesund erscheinenden ungarischen Blutspendern lieferte eine Zöliakieprävalenz, die den Prävalenzwerten aus anderen zentraleuropäischen Ländern bei Erwachsenen gleicht, aber geringer ist als bei Kindern in Ungarn. Global gesehen liegt die Prävalenz der Zöliakie in Ungarn im mittleren Bereich, obwohl die pädiatrischen Daten eine höhere Prävalenz aufzeigen.
Background: Celiac disease (CD) is a common chronic systemic autoimmune disease in Europe. The prevalence of CD in Hungarian children is estimated at 1.2 – 1.4 %. To date, however, no data on adult CD prevalence has been published. Aims: Analysis of the serological evidence for CD among Hungarian adults in order to estimate its prevalence. Methods: Plasma samples from 4155 healthy blood donors were anonymously screened for circulating IgA autoantibodies by a highly sensitive tissue transglutaminase ELISA. Positive results were subsequently confirmed by endomysial antibody test. Results: Endomysial antibody test confirmed positivity in 25 samples suggesting a prevalence of CD of at least 0.6 % (1:166). Since no identification on the samples was provided, no further examinations could be done on endomysial antibody positive individuals. Conclusions: The first serological screening study among healthy Hungarian adult blood donors showed a prevalence of CD similar to other central European countries and lower than that in Hungarian children. Among countries worldwide, the Hungarian prevalence of CD appears to be in the mid-range, although pediatric data suggest a higher prevalence.
Introduction
tant nutrients, i. e., Europe, Western and Northern Asia, parts of America and Australia [1]. Its prevalence in Europe was estimated at 0.15 − 2.7 % [2]. The reason for this wide range is currently unknown, making epidemiological results difficult to evaluate and interpret [2, 3]. Presumably differences in laboratory test methods and criteria set for CD diagnosis as well as genetic, cultural, and
● Gewebstransglutaminase ● Endomysiumantikörper ● Prävalenz ● Zöliakie ● Sprue " " " "
Key words
● tissue transglutaminase ● endomysial antibodies ● prevalence ● celiac disease ● sprue " " " " "
received accepted
18.1.2013 10.4.2013
Bibliography DOI http://dx.doi.org/ 10.1055/s-0033-1335450 Z Gastroenterol 2013; 51: 1235–1239 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0044-2771
Correspondence Dr. Miklós Sárdy Dept. of Dermatology and Allergology, Ludwig Maximilian University Frauenlobstr. 9–11 80337 Munich Germany Tel.: ++ 49/89/51 60 60 10 Fax: ++ 49/89/51 60 61 92 [email protected]
!
Celiac disease (CD, sprue) is a common chronic systemic autoimmune disorder associated with a persistent intolerance to gluten as well as concomitant immune and autoimmune phenomena. It usually affects the white population of countries where wheat, rye, and/or barley are impor-
Sárdy M et al. Celiac Disease Screening … Z Gastroenterol 2013; 51: 1235–1239
Downloaded by: Collections and Technical Services Department. Copyrighted material.
2
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alimentary diversity among the studied geographic areas significantly contribute to the large variations [3]. In addition, frequency variations were observed in the same area during different periods of sampling [4]. Currently the most optimal serological screening method for CD in the healthy population is a quantitative tissue transglutaminase (tTG) ELISA that detects IgA autoantibodies, which is suitable for mass screening due to its high sensitivity and automation potential [3, 5]. Positive or borderline samples may be tested with the more laborious indirect immunofluorescence (IgA endomysial antibody test, EMA), which is more suitable as a confirmatory step due to its higher specificity [3, 5, 6]. The EMA test has proven to be an appropriate screening tool for detecting CD in several epidemiological studies [3, 7 – 13]. In Hungary, the prevalence of CD has only been previously studied in children. In a pilot study at a Hungarian nursery school, it was found in 1 of 85 (1.2 % of 427 children) [14]. This result was later confirmed in a study on a larger population (1.4 % of 2690 preschool children) [15]. The prevalence of CD in Hungarian adults, however, has not been studied. Its determination is important not only for medical-epidemiological but also for health, political, financial, agricultural, commercial, and socio-economic purposes. Such is the reason for performing this study using serological screening methods to evaluate the prevalence of CD among apparently healthy, not-at-risk Hungarian adults.
Study Population and Methods !
Study population Plasma samples were collected in the Hungarian National Blood Transfusion Service, Budapest, from 4155 blood donors on consecutive working days between January 7, 2002 and February 1, 2002. All samples were stored at −78 ºC and assayed between 2002 and 2004. Samples were anonymously examined; data on age and sex were not available. Donors were considered healthy if standard physical examination and laboratory tests (according to the strict regulations for collecting blood samples in the Hungarian National Blood Transfusion Service) revealed no abnormalities; however, these tests did not include any test for CD. The Local Ethical Committee of the Semmelweis University approved the study (registry (TUKEB) number: 113/2001).
escein isothiocyanate-conjugated rabbit anti-human IgA antibodies (1:40 in phosphate-buffered saline; Dako).
Statistics Optical densities (and thus titers given in arbitrary units) did not show a Gaussian distribution in EMA-positive individuals, thus we present both means and medians, and for comparison of optical densities, Mann-Whitney’s non-parametric, unpaired, twotailed test was used. The frequency distribution was tested for normality using the D’Agostino and Pearson omnibus normality test. GraphPad Prism version 4.03 was used for calculations.
Results A total of 4155 healthy blood donors were included in the IgA anti-human tTG ELISA study. An arbitrary cut-off (18 AU) was set. In order to increase the sensitivity of the ELISA safely up to 100 %, the chosen cut-off was lower than both the average titer + 2*standard deviation (19.1 AU) and the 95 % percentile (18.4 AU) after exclusion of EMA positive samples. Thus, positive tTG ELISA results were found in 265 samples (6.4 %) and 3890 samples (93.6 %) tested negative. EMA test was performed only on the tTG ELISA positive samples. " Fig. 1) suggesting a EMA was positive in 25 of the 265 samples (● CD prevalence of 0.6 % (1:166; 95 % CI, 0.41 − 0.89 %). The average and the median of IgA anti-human tTG autoantibody concentrations for all blood donors were 11.7 AU and 10.6 AU, respectively. The frequency distribution of autoantibody concentrations was " Fig. 2). The average and the median of IgA antinot Gaussian (● human tTG autoantibody concentrations for EMA positive blood donors were 64.5 AU and 59.6 AU, respectively. The difference between the titers of EMA negative and positive donors was significant (p < 0.0001). The minimum titer for an EMA positive blood donor was 24.8 AU, i. e., considerably higher than the cut-off and the mean titer+ 2*standard deviation. The maximum titer for an EMA negative blood donor was 48.2 AU. If we accepted the results of EMA tests as diagnostic criterion and chose 24.8 AU as cut-off, then the sensitivity and the specificity of the tTG ELISA
ELISA and EMA tests The IgA anti-human tTG ELISA test was performed using a noncommercial system as described previously [16], the difference being that all samples were tested in duplicate instead of triplicate. The original ELISA test was done in another laboratory with reagents in part from different manufacturers, and it has not been validated for mass screening. Hence, the previously determined cut-off value [16, 17] was not regarded as valid for this study. As the purpose of the ELISA test was merely to preselect samples for the EMA test, the cut-off was deliberately decreased in order to increase the sensitivity safely up to 100 %. In a pilot study, no difference between reactivity in plasma and serum samples was found, thus plasma samples were used (data not shown). In the EMA test, serum IgA antibodies were measured by an indirect immunofluorescence method [18]. All plasma samples were diluted 1:5 in phosphate-buffered saline (pH: 7.4). 10 μm cryostat tissue slides of the aboral part of monkey (Cercopithecinae family) oesophagus were used as antigen. Bound IgA was detected by α-chain specific, fluor-
Sárdy M et al. Celiac Disease Screening … Z Gastroenterol 2013; 51: 1235–1239
Fig. 1 Plasma concentrations of IgA antibodies in the human tissue transglutaminase enzyme-linked immunosorbent assay (tTG ELISA) in healthy appearing blood donors’ samples. The 25 endomysial antibody (EMA)-positive samples are indicated by red hollow squares, the 240 EMAnegative samples by black dots. The cut-off level for positivity (broken line) is drawn at the arbitrary unit (AU) of 18. The 3890 samples having titers below the cut-off are not shown individually and are represented by “3890” below the cut-off line.
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would be 100 % (95 % CI, 86.3 − 100 %) and 99.25 % (95 % CI, 98.9 − 99.5 %), respectively. As all samples were anonymously collected and studied, EMA positive individuals could not be identified and invited for further examinations.
Discussion !
We worked with a non-commercial tTG ELISA for which the cutoff was deliberately decreased in order to increase sensitivity safely up to 100 %. Since a lower specificity was expected, a second diagnostic method was introduced (EMA test) to identify false positive results and to confirm seropositivity [5, 6, 12]. In a recent meta-analysis, 11 studies of high evidence level involving a total of 1034 CD patients were reviewed, and the pooled specificity and sensitivity values of the EMA test were calculated at 98.2 % and ≥ 90 %, respectively [6]. The EMA test, usually performed on monkey oesophagus sections from endangered primates, is laborious and time consuming; it is therefore feasible for testing a few hundred selected sera but not practicable for processing thousands of samples. Since the EMA test is expected to be less sensitive than the tTG ELISA (≥ 90 % versus 100 %), it may have given some false negative results, but not false positive ones. As a consequence, the prevalence we obtained (0.6 %) may probably slightly underestimate the real number of silent CD patients. An overestimation is unlikely but still possible [13]. Assuming that the expected ratio of CD patients is not significantly higher than 2 % in the Hungarian adult population and that the EMA test resulted in less than 10 % false negativity, we can deduce that our test system may have missed ≤ 8 CD patients, thereby potentially raising the prevalence up to 0.8 % (1:126). Extremely high or low CD prevalence data are more likely to be obtained in studies involving less than 1600 individuals for serological testing [2]; therefore, it is safe to say that our data reveals a realistic estimation. Our study is limited by the anonymous plasma samples, hence excluding the possibility of further examination of EMA positive individuals. Thus, verification of the diagnosis with a duodenojejunal biopsy could not be performed. But given that the EMA test has a nearly 100 % positive predictive value [5, 6, 12], the absence of histological verification does not appear to affect our data interpretation significantly. Another limitation was the lack of data on sexual preponderance. Among blood donors in Hungary, males predominate (approx. 60 %) [19], but among CD patients, females may predominate. Between 1975 and 1988, the female:male ratio of registered pediatric CD patients was 1.59 (girls:boys = 81:51) in Budapest (Dr. István
Kósnai, oral communication). In a large study involving 2690 preschool children in Hungary, the gender proportion of verified 32 celiac patients was 3:1 (24 girls:8 boys) [15]. Pediatric data, however, may not necessarily apply to adults. In a very large population study involving 13 145 subjects in the USA, there was no significant difference in sex distribution of adult CD patients [12]. Nevertheless, the prevalence may be slightly underestimated due to a possible disproportion of sex distribution in our study samples. The age of blood donors in Hungary is between 19 and 59 years (mean: 38.2) [19]. Although there is an unexplained difference in the prevalence of CD between adults and children, children cannot be blood donors in Hungary, and no significant difference of CD prevalence among adult age groups could be found [12]. Consequently, the absence of age demographic data is not a major limitation. Finally, although selective IgA deficiency occurs in approx. 2 % of CD patients [20], we performed measurements of total serum IgA only in samples having extremely low titers in the tTG ELISA. No IgA deficiency was found in those samples (data not shown). Compared to other countries, the prevalence of CD in Hungarian " Fig. 3). The difnot-at-risk adults seems to be in the mid-range (● ference in the prevalence of CD between adults and children can " Fig. 3). also be confirmed in Hungary (● In conclusion, we have performed the first serological screening study for CD in healthy Hungarian blood donors, and its prevalence was found to be at least 0.6 %. Although this study was limited by the lack of demographic (age, sex), histological, and total IgA data, we expect the true prevalence of CD for the healthy adult Hungarian population to be within the calculated 95 % CI limits (0.41 − 0.89 %), which is compatible with prevalence data from other central European countries. With regards to the risk for complications of undetected CD, further serological mass screening programmes are justified in the Hungarian population to prevent morbidity and impairment of quality of life.
Abbreviations !
CD CI EMA tTG
celiac disease confidence interval endomysial antibodies tissue transglutaminase.
Sárdy M et al. Celiac Disease Screening … Z Gastroenterol 2013; 51: 1235–1239
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Fig. 2 Frequency distribution of IgA antibody titers in the human tTG ELISA showing a non-Gaussian curve in the healthy blood donors’ samples. AU, arbitrary unit.
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Fig. 3 Prevalence of CD in healthy appearing populations from selected countries compared with Hungary [3, 8 – 12, 15, 21 – 38]. Bars filled with slanting lines show prevalence while the dotted pattern shows the range of prevalence when results of two or more studies are presented. Only studies examining healthy appearing adults using endomysial antibodies and/or duodenojejunal biopsy for diagnosis are presented (if a study also examined children, only the results for adults are shown). The only exception is Hungary (shown in red) as a study in Hungarian children is also presented for comparison [15].
Acknowledgements !
We thank Dr. László Kalász and Dr. Izabella Hoffer (Hungarian National Blood Transfusion Service, Budapest, Hungary) for providing plasma samples from blood donors. We are grateful to Prof. Dr. Mats Paulsson (Institute for Biochemistry II, Medical Faculty, University of Cologne, Cologne, Germany) for the kind gift of recombinant human tTG. We are obliged to Dr. Joyce C. Castillo (Department of Dermatology, Makati Medical Center, Makati, Philippines) for review of the manuscript. We are grateful to Michael Lauseker (Institute for Medical Information Processing, Biometry and Epidemiology, Ludwig Maximilian University, Munich, Germany) for review of the used statistical methods. Further, we are indebted to Dr. István Kósnai (Department of Dermatology, Venereology and Dermatooncology, Semmelweis University, Budapest, Hungary) for his help in the design of the study and Tünde Koltai (Hungarian Coeliac Society, Budapest, Hungary) for her engagement in organization and financial issues. The study was supported by grants from the Hungarian Coeliac Society (Lisztérzékenyek Érdekképviseletének Országos Egyesülete, LÉOE, president: Tünde Koltai) and the Hungarian Medical Scientific Council (ETT 028/2003). Z. K. was a recipient of a scholarship from the Ph. D. Doctoral School of the Semmelweis University, Budapest, Hungary. We report no conflict of interest.
References 01 Corazza GR, Gasbarrini G. Coeliac disease in adults. Baillières Clin Gastroenterol 1995; 9: 329 – 350 02 Dubé C, Rostom A, Sy R et al. The prevalence of celiac disease in averagerisk and at-risk Western European populations: a systematic review. Gastroenterology 2005; 128: S57 – S67 03 Mustalahti K, Catassi C, Reunanen A et al. The prevalence of celiac disease in Europe: results of a centralized, international mass screening project. Ann Med 2010; 42: 587 – 595 04 Lohi S, Mustalahti K, Kaukinen K et al. Increasing prevalence of celiac disease over time. Aliment Pharmacol Ther 2007; 26: 1217 – 1225 05 Biagi F, Pezzimenti D, Campanella J et al. Endomysial and tissue transglutaminase antibodies in coeliac sera: a comparison not influenced by previous serological testing. Scand J Gastroenterol 2001; 36: 955 – 958 06 Giersiepen K, Lelgemann M, Stuhldreher N et al. Accuracy of diagnostic antibody tests for coeliac disease in children: summary of an evidence report. J Pediatr Gastroenterol Nutr 2012; 54: 229 – 241 07 Walker MM, Murray JA, Ronkainen J et al. Detection of celiac disease and lymphocytic enteropathy by parallel serology and histopathology in a population-based study. Gastroenterology 2010; 139: 112 – 119
Sárdy M et al. Celiac Disease Screening … Z Gastroenterol 2013; 51: 1235–1239
08 Kolho KL, Färkkilä MA, Savilahti E. Undiagnosed coeliac disease is common in Finnish adults. Scand J Gastroenterol 1998; 33: 1280 – 1283 09 Pittschieler K, Ladinser B. Coeliac disease: screened by a new strategy. Acta Paediatr Suppl 1996; 412: 42 – 45 10 Trevisiol C, Not T, Berti I et al. Screening for coeliac disease in healthy blood donors at two immuno-transfusion centres in north-east Italy. Ital J Gastroenterol Hepatol 1999; 31: 584 – 586 11 Rostami K, Mulder CJ, Werre JM et al. High prevalence of celiac disease in apparently healthy blood donors suggests a high prevalence of undiagnosed celiac disease in the Dutch population. Scand J Gastroenterol 1999; 34: 276 – 279 12 Fasano A, Berti I, Gerarduzzi T et al. Prevalence of celiac disease in atrisk and not-at-risk groups in the United States: a large multicenter study. Arch Intern Med 2003; 163: 286 – 292 13 Melo SB, Fernandes MI, Peres LC et al. Prevalence and demographic characteristics of celiac disease among blood donors in Ribeirão Preto, State of São Paulo, Brazil. Dig Dis Sci 2006; 51: 1020 – 1025 14 Korponay-Szabó IR, Kovács JB, Czinner A et al. High prevalence of silent celiac disease in preschool children screened with IgA/IgG antiendomysium antibodies. J Pediatr Gastroenterol Nutr 1999; 28: 26 – 30 15 Korponay-Szabó IR, Szabados K, Pusztai J et al. Population screening for coeliac disease in primary care by district nurses using a rapid antibody test: diagnostic accuracy and feasibility study. BMJ 2007; 335: 1244 – 1247 16 Sárdy M, Odenthal U, Kárpáti S et al. Recombinant human tissue transglutaminase ELISA for the diagnosis of gluten sensitive enteropathy. Clin Chem 1999; 45: 2142 – 2149 17 Sárdy M, Csikós M, Geisen C et al. Tissue transglutaminase ELISA positivity in autoimmune disease independent of gluten-sensitive disease. Clin Chim Acta 2007; 376: 126 – 135 18 Collin P, Mäki M, Keyriläinen O et al. Selective IgA deficiency and coeliac disease. Scand J Gastroenterol 1992; 27: 367 – 371 19 Juhász A, Remenyik E, Kónya J et al. Prevalence and age distribution of human herpesvirus-8 specific antibodies in Hungarian blood donors. J Med Virol 2001; 64: 526 – 530 20 Chow MA, Lebwohl B, Reilly NR et al. Immunoglobulin A deficiency in celiac disease. J Clin Gastroenterol 2012; 46: 850 – 854 21 Bahari A, Karimi M, Sanei-Moghaddam I et al. Prevalence of celiac disease among blood donors in Sistan and Balouchestan Province, Southeastern Iran. Arch Iran Med 2010; 13: 301 – 305 22 García Novo MD, Garfia C, Acuña Quirós MD et al. Prevalencia de la enfermedad celiaca en donantes de sangre de la Comunidad de Madrid. Rev Esp Enferm Dig 2007; 99: 337 – 342 23 Henker J, Lösel A, Conrad K et al. Prävalenz der asymptomatischen Zöliakie bei Kindern und Erwachsenen in der Region Dresden. Dtsch Med Wochenschr 2002; 127: 1511 – 1515 24 Hovdenak N, Hovlid E, Aksnes L et al. High prevalence of asymptomatic coeliac disease in Norway: a study of blood donors. Eur J Gastroenterol Hepatol 1999; 11: 185 – 187 25 Ivarsson A, Persson LA, Juto P et al. High prevalence of undiagnosed coeliac disease in adults: a Swedish population-based study. J Intern Med 1999; 245: 63 – 68
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33 Sanders DS, Patel D, Stephenson TJ et al. A primary care cross-sectional study of undiagnosed adult coeliac disease. Eur J Gastroenterol Hepatol 2003; 15: 407 – 413 34 Shahbazkhani B, Malekzadeh R, Sotoudeh M et al. High prevalence of coeliac disease in apparently healthy Iranian blood donors. Eur J Gastroenterol Hepatol 2003; 15: 475 – 478 35 Stroikova M, Augul N, Gureev J et al. Screening of blood donors for tissue transglutaminase antibodies in the Ryazan area (Russia). Dig Liver Dis 2006; 38: 617 – 619 36 Vanciková Z, Chlumecký V, Sokol D et al. The serologic screening for celiac disease in the general population (blood donors) and in some high-risk groups of adults (patients with autoimmune diseases, osteoporosis and infertility) in the Czech Republic. Folia Microbiol (Praha) 2002; 47: 753 – 758 37 Weile I, Grodzinsky E, Skogh T et al. High prevalence rates of adult silent coeliac disease, as seen in Sweden, must be expected in Denmark. APMIS 2001; 109: 745 – 750 38 West J, Logan RF, Hill PG et al. Seroprevalence, correlates, and characteristics of undetected coeliac disease in England. Gut 2003; 52: 960 – 965
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26 Johannsson GF, Kristjansson G, Cariglia N et al. The prevalence of celiac disease in blood donors in Iceland. Dig Dis Sci 2009; 54: 348 – 350 27 Johnston SD, Watson RG, McMillan SA et al. Prevalence of coeliac disease in Northern Ireland. Lancet 1997; 350: 1370 28 Kochhar R, Sachdev S, Kochhar R et al. Prevalence of coeliac disease in healthy blood donors: a study from north India. Dig Liver Dis 2012; 44: 530 – 532 29 Not T, Horvath K, Hill ID et al. Celiac disease risk in the USA: high prevalence of antiendomysium antibodies in healthy blood donors. Scand J Gastroenterol 1998; 33: 494 – 498 30 Oliveira RP, Sdepanian VL, Barreto JA et al. High prevalence of celiac disease in Brazilian blood donor volunteers based on screening by IgA antitissue transglutaminase antibody. Eur J Gastroenterol Hepatol 2007; 19: 43 – 49 31 Pratesi R, Gandolfi L, Garcia SG et al. Prevalence of coeliac disease: unexplained age-related variation in the same population. Scand J Gastroenterol 2003; 38: 747 – 750 32 Riestra S, Fernández E, Rodrigo L et al. Prevalence of coeliac disease in the general population of northern Spain. Strategies of serologic screening. Scand J Gastroenterol 2000; 35: 398 – 402
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Celiac Disease Screening among Healthy Blood Donors in Hungary Zöliakie-Screening bei gesunden Blutspendern in Ungarn Authors
M. Sárdy1, 2, Z. Kornseé1, D. Kelemen1, S. Papp1, M. Medvecz1, S. Kárpáti1
Affiliations
1
Department of Dermatology, Venereology and Dermatooncology, Semmelweis University, Budapest Department of Dermatology and Allergology, Ludwig Maximilian University, Munich, Germany
Schlüsselwörter
Zusammenfassung
Abstract
"
!
!
Hintergrund: Die Zöliakie ist eine häufige, chronische systemische Autoimmunerkrankung in Europa. Ihre Prävalenz bei Kindern in der Allgemeinbevölkerung Ungarns liegt bei 1,2 – 1,4 %. Bislang gibt es keine Daten hinsichtlich der Prävalenz bei Erwachsenen in Ungarn. Ziele: Analyse der Seren von Erwachsenen in Ungarn zur Ermittlung der Prävalenz der Zöliakie. Methoden: Plasmaproben von 4155 gesund erscheinenden Blutspendern wurden anonymisiert und hinsichtlich zirkulierender IgA-Autoantikörper gegen Gewebstransglutaminase mittels eines hoch sensitiven ELISA untersucht. Die positiven Ergebnisse wurden mithilfe eines EndomysiumAntikörpertests verifiziert. Ergebnisse: Der Endomysium-Antikörpertest war bei 25 Proben positiv, insofern scheint die Prävalenz der Zöliakie bei mindestens etwa 0,6 % (1:166) zu liegen. Da die Proben anonymisiert getestet wurden, konnten die Betroffenen nicht weiter untersucht werden. Schlussfolgerungen: Die erste serologische Screeningstudie bei gesund erscheinenden ungarischen Blutspendern lieferte eine Zöliakieprävalenz, die den Prävalenzwerten aus anderen zentraleuropäischen Ländern bei Erwachsenen gleicht, aber geringer ist als bei Kindern in Ungarn. Global gesehen liegt die Prävalenz der Zöliakie in Ungarn im mittleren Bereich, obwohl die pädiatrischen Daten eine höhere Prävalenz aufzeigen.
Background: Celiac disease (CD) is a common chronic systemic autoimmune disease in Europe. The prevalence of CD in Hungarian children is estimated at 1.2 – 1.4 %. To date, however, no data on adult CD prevalence has been published. Aims: Analysis of the serological evidence for CD among Hungarian adults in order to estimate its prevalence. Methods: Plasma samples from 4155 healthy blood donors were anonymously screened for circulating IgA autoantibodies by a highly sensitive tissue transglutaminase ELISA. Positive results were subsequently confirmed by endomysial antibody test. Results: Endomysial antibody test confirmed positivity in 25 samples suggesting a prevalence of CD of at least 0.6 % (1:166). Since no identification on the samples was provided, no further examinations could be done on endomysial antibody positive individuals. Conclusions: The first serological screening study among healthy Hungarian adult blood donors showed a prevalence of CD similar to other central European countries and lower than that in Hungarian children. Among countries worldwide, the Hungarian prevalence of CD appears to be in the mid-range, although pediatric data suggest a higher prevalence.
Introduction
tant nutrients, i. e., Europe, Western and Northern Asia, parts of America and Australia [1]. Its prevalence in Europe was estimated at 0.15 − 2.7 % [2]. The reason for this wide range is currently unknown, making epidemiological results difficult to evaluate and interpret [2, 3]. Presumably differences in laboratory test methods and criteria set for CD diagnosis as well as genetic, cultural, and
● Gewebstransglutaminase ● Endomysiumantikörper ● Prävalenz ● Zöliakie ● Sprue " " " "
Key words
● tissue transglutaminase ● endomysial antibodies ● prevalence ● celiac disease ● sprue " " " " "
received accepted
18.1.2013 10.4.2013
Bibliography DOI http://dx.doi.org/ 10.1055/s-0033-1335450 Z Gastroenterol 2013; 51: 1235–1239 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0044-2771
Correspondence Dr. Miklós Sárdy Dept. of Dermatology and Allergology, Ludwig Maximilian University Frauenlobstr. 9–11 80337 Munich Germany Tel.: ++ 49/89/51 60 60 10 Fax: ++ 49/89/51 60 61 92 [email protected]
!
Celiac disease (CD, sprue) is a common chronic systemic autoimmune disorder associated with a persistent intolerance to gluten as well as concomitant immune and autoimmune phenomena. It usually affects the white population of countries where wheat, rye, and/or barley are impor-
Sárdy M et al. Celiac Disease Screening … Z Gastroenterol 2013; 51: 1235–1239
Downloaded by: Collections and Technical Services Department. Copyrighted material.
2
Originalarbeit
alimentary diversity among the studied geographic areas significantly contribute to the large variations [3]. In addition, frequency variations were observed in the same area during different periods of sampling [4]. Currently the most optimal serological screening method for CD in the healthy population is a quantitative tissue transglutaminase (tTG) ELISA that detects IgA autoantibodies, which is suitable for mass screening due to its high sensitivity and automation potential [3, 5]. Positive or borderline samples may be tested with the more laborious indirect immunofluorescence (IgA endomysial antibody test, EMA), which is more suitable as a confirmatory step due to its higher specificity [3, 5, 6]. The EMA test has proven to be an appropriate screening tool for detecting CD in several epidemiological studies [3, 7 – 13]. In Hungary, the prevalence of CD has only been previously studied in children. In a pilot study at a Hungarian nursery school, it was found in 1 of 85 (1.2 % of 427 children) [14]. This result was later confirmed in a study on a larger population (1.4 % of 2690 preschool children) [15]. The prevalence of CD in Hungarian adults, however, has not been studied. Its determination is important not only for medical-epidemiological but also for health, political, financial, agricultural, commercial, and socio-economic purposes. Such is the reason for performing this study using serological screening methods to evaluate the prevalence of CD among apparently healthy, not-at-risk Hungarian adults.
Study Population and Methods !
Study population Plasma samples were collected in the Hungarian National Blood Transfusion Service, Budapest, from 4155 blood donors on consecutive working days between January 7, 2002 and February 1, 2002. All samples were stored at −78 ºC and assayed between 2002 and 2004. Samples were anonymously examined; data on age and sex were not available. Donors were considered healthy if standard physical examination and laboratory tests (according to the strict regulations for collecting blood samples in the Hungarian National Blood Transfusion Service) revealed no abnormalities; however, these tests did not include any test for CD. The Local Ethical Committee of the Semmelweis University approved the study (registry (TUKEB) number: 113/2001).
escein isothiocyanate-conjugated rabbit anti-human IgA antibodies (1:40 in phosphate-buffered saline; Dako).
Statistics Optical densities (and thus titers given in arbitrary units) did not show a Gaussian distribution in EMA-positive individuals, thus we present both means and medians, and for comparison of optical densities, Mann-Whitney’s non-parametric, unpaired, twotailed test was used. The frequency distribution was tested for normality using the D’Agostino and Pearson omnibus normality test. GraphPad Prism version 4.03 was used for calculations.
Results A total of 4155 healthy blood donors were included in the IgA anti-human tTG ELISA study. An arbitrary cut-off (18 AU) was set. In order to increase the sensitivity of the ELISA safely up to 100 %, the chosen cut-off was lower than both the average titer + 2*standard deviation (19.1 AU) and the 95 % percentile (18.4 AU) after exclusion of EMA positive samples. Thus, positive tTG ELISA results were found in 265 samples (6.4 %) and 3890 samples (93.6 %) tested negative. EMA test was performed only on the tTG ELISA positive samples. " Fig. 1) suggesting a EMA was positive in 25 of the 265 samples (● CD prevalence of 0.6 % (1:166; 95 % CI, 0.41 − 0.89 %). The average and the median of IgA anti-human tTG autoantibody concentrations for all blood donors were 11.7 AU and 10.6 AU, respectively. The frequency distribution of autoantibody concentrations was " Fig. 2). The average and the median of IgA antinot Gaussian (● human tTG autoantibody concentrations for EMA positive blood donors were 64.5 AU and 59.6 AU, respectively. The difference between the titers of EMA negative and positive donors was significant (p < 0.0001). The minimum titer for an EMA positive blood donor was 24.8 AU, i. e., considerably higher than the cut-off and the mean titer+ 2*standard deviation. The maximum titer for an EMA negative blood donor was 48.2 AU. If we accepted the results of EMA tests as diagnostic criterion and chose 24.8 AU as cut-off, then the sensitivity and the specificity of the tTG ELISA
ELISA and EMA tests The IgA anti-human tTG ELISA test was performed using a noncommercial system as described previously [16], the difference being that all samples were tested in duplicate instead of triplicate. The original ELISA test was done in another laboratory with reagents in part from different manufacturers, and it has not been validated for mass screening. Hence, the previously determined cut-off value [16, 17] was not regarded as valid for this study. As the purpose of the ELISA test was merely to preselect samples for the EMA test, the cut-off was deliberately decreased in order to increase the sensitivity safely up to 100 %. In a pilot study, no difference between reactivity in plasma and serum samples was found, thus plasma samples were used (data not shown). In the EMA test, serum IgA antibodies were measured by an indirect immunofluorescence method [18]. All plasma samples were diluted 1:5 in phosphate-buffered saline (pH: 7.4). 10 μm cryostat tissue slides of the aboral part of monkey (Cercopithecinae family) oesophagus were used as antigen. Bound IgA was detected by α-chain specific, fluor-
Sárdy M et al. Celiac Disease Screening … Z Gastroenterol 2013; 51: 1235–1239
Fig. 1 Plasma concentrations of IgA antibodies in the human tissue transglutaminase enzyme-linked immunosorbent assay (tTG ELISA) in healthy appearing blood donors’ samples. The 25 endomysial antibody (EMA)-positive samples are indicated by red hollow squares, the 240 EMAnegative samples by black dots. The cut-off level for positivity (broken line) is drawn at the arbitrary unit (AU) of 18. The 3890 samples having titers below the cut-off are not shown individually and are represented by “3890” below the cut-off line.
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would be 100 % (95 % CI, 86.3 − 100 %) and 99.25 % (95 % CI, 98.9 − 99.5 %), respectively. As all samples were anonymously collected and studied, EMA positive individuals could not be identified and invited for further examinations.
Discussion !
We worked with a non-commercial tTG ELISA for which the cutoff was deliberately decreased in order to increase sensitivity safely up to 100 %. Since a lower specificity was expected, a second diagnostic method was introduced (EMA test) to identify false positive results and to confirm seropositivity [5, 6, 12]. In a recent meta-analysis, 11 studies of high evidence level involving a total of 1034 CD patients were reviewed, and the pooled specificity and sensitivity values of the EMA test were calculated at 98.2 % and ≥ 90 %, respectively [6]. The EMA test, usually performed on monkey oesophagus sections from endangered primates, is laborious and time consuming; it is therefore feasible for testing a few hundred selected sera but not practicable for processing thousands of samples. Since the EMA test is expected to be less sensitive than the tTG ELISA (≥ 90 % versus 100 %), it may have given some false negative results, but not false positive ones. As a consequence, the prevalence we obtained (0.6 %) may probably slightly underestimate the real number of silent CD patients. An overestimation is unlikely but still possible [13]. Assuming that the expected ratio of CD patients is not significantly higher than 2 % in the Hungarian adult population and that the EMA test resulted in less than 10 % false negativity, we can deduce that our test system may have missed ≤ 8 CD patients, thereby potentially raising the prevalence up to 0.8 % (1:126). Extremely high or low CD prevalence data are more likely to be obtained in studies involving less than 1600 individuals for serological testing [2]; therefore, it is safe to say that our data reveals a realistic estimation. Our study is limited by the anonymous plasma samples, hence excluding the possibility of further examination of EMA positive individuals. Thus, verification of the diagnosis with a duodenojejunal biopsy could not be performed. But given that the EMA test has a nearly 100 % positive predictive value [5, 6, 12], the absence of histological verification does not appear to affect our data interpretation significantly. Another limitation was the lack of data on sexual preponderance. Among blood donors in Hungary, males predominate (approx. 60 %) [19], but among CD patients, females may predominate. Between 1975 and 1988, the female:male ratio of registered pediatric CD patients was 1.59 (girls:boys = 81:51) in Budapest (Dr. István
Kósnai, oral communication). In a large study involving 2690 preschool children in Hungary, the gender proportion of verified 32 celiac patients was 3:1 (24 girls:8 boys) [15]. Pediatric data, however, may not necessarily apply to adults. In a very large population study involving 13 145 subjects in the USA, there was no significant difference in sex distribution of adult CD patients [12]. Nevertheless, the prevalence may be slightly underestimated due to a possible disproportion of sex distribution in our study samples. The age of blood donors in Hungary is between 19 and 59 years (mean: 38.2) [19]. Although there is an unexplained difference in the prevalence of CD between adults and children, children cannot be blood donors in Hungary, and no significant difference of CD prevalence among adult age groups could be found [12]. Consequently, the absence of age demographic data is not a major limitation. Finally, although selective IgA deficiency occurs in approx. 2 % of CD patients [20], we performed measurements of total serum IgA only in samples having extremely low titers in the tTG ELISA. No IgA deficiency was found in those samples (data not shown). Compared to other countries, the prevalence of CD in Hungarian " Fig. 3). The difnot-at-risk adults seems to be in the mid-range (● ference in the prevalence of CD between adults and children can " Fig. 3). also be confirmed in Hungary (● In conclusion, we have performed the first serological screening study for CD in healthy Hungarian blood donors, and its prevalence was found to be at least 0.6 %. Although this study was limited by the lack of demographic (age, sex), histological, and total IgA data, we expect the true prevalence of CD for the healthy adult Hungarian population to be within the calculated 95 % CI limits (0.41 − 0.89 %), which is compatible with prevalence data from other central European countries. With regards to the risk for complications of undetected CD, further serological mass screening programmes are justified in the Hungarian population to prevent morbidity and impairment of quality of life.
Abbreviations !
CD CI EMA tTG
celiac disease confidence interval endomysial antibodies tissue transglutaminase.
Sárdy M et al. Celiac Disease Screening … Z Gastroenterol 2013; 51: 1235–1239
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Fig. 2 Frequency distribution of IgA antibody titers in the human tTG ELISA showing a non-Gaussian curve in the healthy blood donors’ samples. AU, arbitrary unit.
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Fig. 3 Prevalence of CD in healthy appearing populations from selected countries compared with Hungary [3, 8 – 12, 15, 21 – 38]. Bars filled with slanting lines show prevalence while the dotted pattern shows the range of prevalence when results of two or more studies are presented. Only studies examining healthy appearing adults using endomysial antibodies and/or duodenojejunal biopsy for diagnosis are presented (if a study also examined children, only the results for adults are shown). The only exception is Hungary (shown in red) as a study in Hungarian children is also presented for comparison [15].
Acknowledgements !
We thank Dr. László Kalász and Dr. Izabella Hoffer (Hungarian National Blood Transfusion Service, Budapest, Hungary) for providing plasma samples from blood donors. We are grateful to Prof. Dr. Mats Paulsson (Institute for Biochemistry II, Medical Faculty, University of Cologne, Cologne, Germany) for the kind gift of recombinant human tTG. We are obliged to Dr. Joyce C. Castillo (Department of Dermatology, Makati Medical Center, Makati, Philippines) for review of the manuscript. We are grateful to Michael Lauseker (Institute for Medical Information Processing, Biometry and Epidemiology, Ludwig Maximilian University, Munich, Germany) for review of the used statistical methods. Further, we are indebted to Dr. István Kósnai (Department of Dermatology, Venereology and Dermatooncology, Semmelweis University, Budapest, Hungary) for his help in the design of the study and Tünde Koltai (Hungarian Coeliac Society, Budapest, Hungary) for her engagement in organization and financial issues. The study was supported by grants from the Hungarian Coeliac Society (Lisztérzékenyek Érdekképviseletének Országos Egyesülete, LÉOE, president: Tünde Koltai) and the Hungarian Medical Scientific Council (ETT 028/2003). Z. K. was a recipient of a scholarship from the Ph. D. Doctoral School of the Semmelweis University, Budapest, Hungary. We report no conflict of interest.
References 01 Corazza GR, Gasbarrini G. Coeliac disease in adults. Baillières Clin Gastroenterol 1995; 9: 329 – 350 02 Dubé C, Rostom A, Sy R et al. The prevalence of celiac disease in averagerisk and at-risk Western European populations: a systematic review. Gastroenterology 2005; 128: S57 – S67 03 Mustalahti K, Catassi C, Reunanen A et al. The prevalence of celiac disease in Europe: results of a centralized, international mass screening project. Ann Med 2010; 42: 587 – 595 04 Lohi S, Mustalahti K, Kaukinen K et al. Increasing prevalence of celiac disease over time. Aliment Pharmacol Ther 2007; 26: 1217 – 1225 05 Biagi F, Pezzimenti D, Campanella J et al. Endomysial and tissue transglutaminase antibodies in coeliac sera: a comparison not influenced by previous serological testing. Scand J Gastroenterol 2001; 36: 955 – 958 06 Giersiepen K, Lelgemann M, Stuhldreher N et al. Accuracy of diagnostic antibody tests for coeliac disease in children: summary of an evidence report. J Pediatr Gastroenterol Nutr 2012; 54: 229 – 241 07 Walker MM, Murray JA, Ronkainen J et al. Detection of celiac disease and lymphocytic enteropathy by parallel serology and histopathology in a population-based study. Gastroenterology 2010; 139: 112 – 119
Sárdy M et al. Celiac Disease Screening … Z Gastroenterol 2013; 51: 1235–1239
08 Kolho KL, Färkkilä MA, Savilahti E. Undiagnosed coeliac disease is common in Finnish adults. Scand J Gastroenterol 1998; 33: 1280 – 1283 09 Pittschieler K, Ladinser B. Coeliac disease: screened by a new strategy. Acta Paediatr Suppl 1996; 412: 42 – 45 10 Trevisiol C, Not T, Berti I et al. Screening for coeliac disease in healthy blood donors at two immuno-transfusion centres in north-east Italy. Ital J Gastroenterol Hepatol 1999; 31: 584 – 586 11 Rostami K, Mulder CJ, Werre JM et al. High prevalence of celiac disease in apparently healthy blood donors suggests a high prevalence of undiagnosed celiac disease in the Dutch population. Scand J Gastroenterol 1999; 34: 276 – 279 12 Fasano A, Berti I, Gerarduzzi T et al. Prevalence of celiac disease in atrisk and not-at-risk groups in the United States: a large multicenter study. Arch Intern Med 2003; 163: 286 – 292 13 Melo SB, Fernandes MI, Peres LC et al. Prevalence and demographic characteristics of celiac disease among blood donors in Ribeirão Preto, State of São Paulo, Brazil. Dig Dis Sci 2006; 51: 1020 – 1025 14 Korponay-Szabó IR, Kovács JB, Czinner A et al. High prevalence of silent celiac disease in preschool children screened with IgA/IgG antiendomysium antibodies. J Pediatr Gastroenterol Nutr 1999; 28: 26 – 30 15 Korponay-Szabó IR, Szabados K, Pusztai J et al. Population screening for coeliac disease in primary care by district nurses using a rapid antibody test: diagnostic accuracy and feasibility study. BMJ 2007; 335: 1244 – 1247 16 Sárdy M, Odenthal U, Kárpáti S et al. Recombinant human tissue transglutaminase ELISA for the diagnosis of gluten sensitive enteropathy. Clin Chem 1999; 45: 2142 – 2149 17 Sárdy M, Csikós M, Geisen C et al. Tissue transglutaminase ELISA positivity in autoimmune disease independent of gluten-sensitive disease. Clin Chim Acta 2007; 376: 126 – 135 18 Collin P, Mäki M, Keyriläinen O et al. Selective IgA deficiency and coeliac disease. Scand J Gastroenterol 1992; 27: 367 – 371 19 Juhász A, Remenyik E, Kónya J et al. Prevalence and age distribution of human herpesvirus-8 specific antibodies in Hungarian blood donors. J Med Virol 2001; 64: 526 – 530 20 Chow MA, Lebwohl B, Reilly NR et al. Immunoglobulin A deficiency in celiac disease. J Clin Gastroenterol 2012; 46: 850 – 854 21 Bahari A, Karimi M, Sanei-Moghaddam I et al. Prevalence of celiac disease among blood donors in Sistan and Balouchestan Province, Southeastern Iran. Arch Iran Med 2010; 13: 301 – 305 22 García Novo MD, Garfia C, Acuña Quirós MD et al. Prevalencia de la enfermedad celiaca en donantes de sangre de la Comunidad de Madrid. Rev Esp Enferm Dig 2007; 99: 337 – 342 23 Henker J, Lösel A, Conrad K et al. Prävalenz der asymptomatischen Zöliakie bei Kindern und Erwachsenen in der Region Dresden. Dtsch Med Wochenschr 2002; 127: 1511 – 1515 24 Hovdenak N, Hovlid E, Aksnes L et al. High prevalence of asymptomatic coeliac disease in Norway: a study of blood donors. Eur J Gastroenterol Hepatol 1999; 11: 185 – 187 25 Ivarsson A, Persson LA, Juto P et al. High prevalence of undiagnosed coeliac disease in adults: a Swedish population-based study. J Intern Med 1999; 245: 63 – 68
Downloaded by: Collections and Technical Services Department. Copyrighted material.
1238
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33 Sanders DS, Patel D, Stephenson TJ et al. A primary care cross-sectional study of undiagnosed adult coeliac disease. Eur J Gastroenterol Hepatol 2003; 15: 407 – 413 34 Shahbazkhani B, Malekzadeh R, Sotoudeh M et al. High prevalence of coeliac disease in apparently healthy Iranian blood donors. Eur J Gastroenterol Hepatol 2003; 15: 475 – 478 35 Stroikova M, Augul N, Gureev J et al. Screening of blood donors for tissue transglutaminase antibodies in the Ryazan area (Russia). Dig Liver Dis 2006; 38: 617 – 619 36 Vanciková Z, Chlumecký V, Sokol D et al. The serologic screening for celiac disease in the general population (blood donors) and in some high-risk groups of adults (patients with autoimmune diseases, osteoporosis and infertility) in the Czech Republic. Folia Microbiol (Praha) 2002; 47: 753 – 758 37 Weile I, Grodzinsky E, Skogh T et al. High prevalence rates of adult silent coeliac disease, as seen in Sweden, must be expected in Denmark. APMIS 2001; 109: 745 – 750 38 West J, Logan RF, Hill PG et al. Seroprevalence, correlates, and characteristics of undetected coeliac disease in England. Gut 2003; 52: 960 – 965
Downloaded by: Collections and Technical Services Department. Copyrighted material.
26 Johannsson GF, Kristjansson G, Cariglia N et al. The prevalence of celiac disease in blood donors in Iceland. Dig Dis Sci 2009; 54: 348 – 350 27 Johnston SD, Watson RG, McMillan SA et al. Prevalence of coeliac disease in Northern Ireland. Lancet 1997; 350: 1370 28 Kochhar R, Sachdev S, Kochhar R et al. Prevalence of coeliac disease in healthy blood donors: a study from north India. Dig Liver Dis 2012; 44: 530 – 532 29 Not T, Horvath K, Hill ID et al. Celiac disease risk in the USA: high prevalence of antiendomysium antibodies in healthy blood donors. Scand J Gastroenterol 1998; 33: 494 – 498 30 Oliveira RP, Sdepanian VL, Barreto JA et al. High prevalence of celiac disease in Brazilian blood donor volunteers based on screening by IgA antitissue transglutaminase antibody. Eur J Gastroenterol Hepatol 2007; 19: 43 – 49 31 Pratesi R, Gandolfi L, Garcia SG et al. Prevalence of coeliac disease: unexplained age-related variation in the same population. Scand J Gastroenterol 2003; 38: 747 – 750 32 Riestra S, Fernández E, Rodrigo L et al. Prevalence of coeliac disease in the general population of northern Spain. Strategies of serologic screening. Scand J Gastroenterol 2000; 35: 398 – 402
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