http://informahealthcare.com/erc ISSN: 0743-5800 (print), 1532-4206 (electronic) Endocr Res, Early Online: 1–4 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/07435800.2015.1015727
The relationship between cytotoxin-associated gene A positive Helicobacter pylori infection and autoimmune thyroid disease Muyesser Sayki Arslan1, Fuat Ekiz2, Murat Deveci2, Mustafa Sahin3, Oya Topaloglu1, Basak Karbek1, Esra Tutal1, Zeynep Ginis4, Erman Cakal1, Mustafa Ozbek1, Osman Yuksel2, and Tuncay Delibasi1 Department of Endocrinology and Metabolic Diseases and 2Department of Gastroenterology, Diskapi Yildirim Beyazit Training and Research Hospital, Ankara, Turkey, 3Department of Endocrinology and Metabolism, School of Medicine, Ankara University, Ankara, Turkey, and 4Department of Clinical Biochemistry, Diskapi Yildirim Beyazit Training and Research Hospital, Ankara, Turkey
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1
Abstract
Keywords
Objective: The aim of this study was to determine whether there is an association between cagA [cytotoxin-associated gene A] positivity and thyroid autoimmunity and thyroid volume. Methods: This prospective study included 78 Helicobacter pylori-positive (H. pylori) dyspeptic patients in the study group, and 50 age-, gender-, and body mass index-matched H. pylorinegative dyspeptic patients in the control group. All the controls were evaluated via upper gastrointestinal endoscopic biopsy or breath test, and were found as H. pylori negative. Gastric biopsy specimens were obtained via endoscopy and histological examination was performed for documentation of H. pylori. Results: In all, 55.1% (n ¼ 43) of the H. pylori-positive patients were cagA positive. There was no significant difference in metabolic syndrome parameters or thyroid function test results between the study and control groups. The frequency of anti-TPO and Hashimoto’s thyroiditis positivity was significantly higher in the study group than in the control group. Thyroid volume was higher and severe parenchymal heterogeneity was more common in the H. pylori-positive patients. Conclusions: H. pylori infection might be a risk factor for autoimmune thyroid disease and high thyroid volume in patients diagnosed with histological evaluation. However, cagA positivity has no additional effect on these parameters.
Helicobacter pylori, cytotoxin-associated gene A, thyroid function test
Introduction Helicobacter pylori (H. pylori) infection is the most common infectious disease and infects approximately 50% of the world’s population (1). Conditions characteristically associated with this infection are chronic gastritis, peptic ulcer disease, and gastric malignancies (2,3). When H. pylori infection is not adequately treated, it can cause extra-digestive disorders via local production and systemic diffusion of proinflammatory cytokines (4). Expression of the H. pylori protein cytotoxin-associated gene A (cagA) indicates the existence of a virulent H. pylori strain (5). It is reported that the presence of cagA protein is a risk factor for acne rosacea, stroke, and coronary heart diseases (6). Endocrine disorders thought to be associated with H. pylori infection include diabetes mellitus, autoimmune thyroid disease (ATD), dyslipidemia, obesity, primary hyperparathyroidism and osteoporosis (7). Several studies investigated the relationship between H. pylori infection and ATD, but the findings are
Correspondence: Muyesser Sayki Arslan, Department of Endocrinology and Metabolic Diseases, Diskapi Yildirim Beyazit Training and Research Hospital, Ankara 06110, Turkey. E-mail: [email protected]
History Received 16 June 2014 Revised 8 November 2014 Accepted 1 February 2015 Published online 30 March 2015
inconsistent. Bassi et al. investigated the correlation between the prevalence of H. pylori infection and ATD, and reported that H. pylori infection is correlated with Graves’ disease (8,9); however, other studies did not observe similar association (10,11). Current data shows that there is a potential association between insulin resistance (IR) and H. pylori infection; however, additional research is required to clarify the degree to which it affects IR (12). Although the pathophysiology of this relationship is not fully understood, an increase in the fetuin A level, and the release of vasoactive and proinflammatory substances are some of the main factors that contribute to IR (13,14). It is possible that H. pylori infection affects the thyroid via autoimmunity caused by molecular mimicry (15). It was reported that monoclonal antibodies to an H. pylori organism with cagA positivity reacted with follicular cells of the thyroid gland, and that an H. pylori strain possessing the cagA pathogenicity island has a gene encoding for an endogenous peroxidase (16). As ATD and IR are the most commonly diagnosed endocrine disorders, the present study was undertaken with the aim to determine if there is an association between cagA positivity, and Hashimoto’s thyroiditis and thyroid volume. We also evaluated metabolic parameters such as fasting glucose and IR that is known to affect thyroid volume (17).
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Materials and methods This study was conducted at the Diskapi Yildirim Beyazit Training and Research Hospital in Ankara, Turkey. Participants were referred from the gastroenterology outpatient clinic to the endocrinology, diabetes and metabolism outpatient clinic. Seventy-eight patients in the study group were selected from those with gastrointestinal system symptoms evaluated by upper gastrointestinal (GI) endoscopy. All patients were histopathologically positive for H. pylori infection. None of the patients had any comorbidity, such as hypertension, diabetes, cardiovascular disorders, renal insufficiency, hepatic diseases, or thyroid dysfunction, and none were using any medications. The control group included 50 H. pylori-negative healthy volunteers. Helicobacter pylori negativity was determined by histopathological evaluation in 32 of the controls and by non-invasive C14 urea-breath test in 18 of the controls. Blood samples were collected from all of the participants and centrifuged. Serum was stored at 80 C in aliquots until the day of analysis. Serum H. pylori IgG antibodies to the cagA level were measured using a solidphase enzyme-linked immunosorbent assay (ELISA), according to the manufacturer’s instructions (DRG International, Inc., Springfield, NJ). Absorbance at 450 nm was measured using an ELISA reader (EPOCH). The ATD diagnosis was made to determine whether the patient had positivity of thyroid autoantibody (anti-Tg and anti-TPO) positivity and at least one of the criteria of thyroid parenchymal heterogeneity according to thyroid ultrasonography (USG), abnormality in thyroid function tests and diffuse goiter with physical examination (18). Following overnight fasting, venous blood samples for biochemical evaluation including routine renal and hepatic function, fasting glucose, insulin, high density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and triglyceride levels were taken from each participant. Thyroid function tests were assessed via measurement of FT4 and TSH, along with anti-thyroid peroxidase (anti-TPO) and anti-thyroglobulin (anti-Tg) using immunochemiluminescent assays (Immulite 2000; Diagnostic Products Corp., Los Angeles, CA) with the following normal ranges: TSH: 0.55–4.78 mIU/mL–1; FT4: 0.74– 1.52 ng/dL–1; anti-TPO: 0–57 IU/mL–1; anti-Tg: 564 IU/mL–1. IR was diagnosed based on the homeostasis model assessment (HOMA) index in all participants (19). The same physician measured each participant’s height, weight and waist circumference (WC). WC (in centimeters cm) was measured on a horizontal plane at the level of the umbilicus. The body mass index (BMI) was calculated by dividing body weight (kg) by height in square centimeters (cm2). Thyroid USG was performed in each participant by the same physician using a 12-MHz linear probe (Hitachi EUB 7000 HV, Tokyo, Japan). Thyroid gland volume was calculated according to the ellipsoid formula: volume (mL) ¼ length (cm) width (cm) depth (cm) /6 (9). Fine needle aspiration biopsy of the thyroid (FNAB) was performed in all thyroid nodules greater than 1 cm. Data were analyzed using SPSS v.18.0 for Windows (SPSS Inc., Chicago, IL). The normality of the distribution of continuous variables was determined using the
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Kolmogorov-Smirnov test. The Mann-Whitney test was used to compare variables that were not normally distributed and Student’s t test was used to compare variables with normal distribution. Categorical features and relationships between groups were assessed using an appropriate chi-square test. The level of statistical significance was set as p50.05.
Results Demographic, biochemical and thyroid volume details of the patients are shown in Table 1. As illustrated, the study included 128 volunteers (78 in the study group and 50 in the control group). The groups were matched for age, gender and BMI. The mean fasting blood glucose level and WC in both groups were similar. In addition, there wasn’t a significant difference in the mean LDL-C level or HOMA-IR between the two groups (Table 1). TSH levels were higher in the study group, but the difference was not significant. FT4 and FT3 levels were similar in both groups. The anti-TPO positivity rate was higher in the study group (p50.05), whereas the anti-Tg positivity rate was similar in both groups. Mean thyroid volume did not differ between the two groups. Moreover, nodule volume was higher in the study group than in the control group (p ¼ 0.05). We also evaluated the prevalence of positivity of ATD, and it was significantly higher in patients than in controls (70%, n ¼ 55 versus 30%, n ¼ 23; p ¼ 0.02, respectively). However, there was no significant difference in terms of HT prevalence according to cagA status. Fine needle thyroid aspiration biopsy was performed in all patients with risk factors and the results were reported as benign in all of them. The percentage of patients with thyroid nodules was similar in both groups (32.5 versus 26.5%; p ¼ 0.474). Mild, moderate, and severe thyroid heterogeneity was observed in 35, 21.3, and 18.8% of the study group, versus 24.5, 34.7 and 4.1% of the control group, respectively, Table 1. Demographics, biochemical characteristics, and thyroid volume in the H. pylori-positive and -negative patients.
Male/female Age (years) BMI (kg m2) WC (cm) FBG (mg dL1) HDL-C (mg dL1) LDL-C (mg dL1) TG (mg dL1) Fasting insulin (IU mL1) HOMA-IR (%) TSH (mIU/mL1) FT4 (ng dL1) Anti-TPO positivity (n, %) Anti-Tg positivity (n, %) Thyroid volume (mL) Nodule volume (mL)
H. pylori (+) (n ¼ 78)
H. pylori (–) (n ¼ 50)
p
24/54 40.9 ± 11.8 27.8 ± 7.5 89.6 ± 10.1 89.7 ± 11.6 45.5 ± 11.8 112.5 ± 31.7 118.4 ± 51.1 8.6 ± 5.8 1.9 ± 1.3 2.0 ± 1.7 1.0 ± 0.17 46 (58,2%) 22 (27,8%) 12.3 ± 7.0 0.9 ± 2.1
17/33 36.9 ± 11.8 25.9 ± 4.2 86.1 ± 8.2 86.5 ± 7.9 47.0 ± 10.7 111.0 ± 21.5 130.6 ± 65.4 9.2 ± 3.5 1.9 ± 0.8 1.9 ± 1.1 1.0 ± 0.3 33 (41,8) 12 (%24) 11.3 ± 4.1 0.11 ± 0.1
0.709 0.065 0.254 0.130 0.060 0.468 0.773 0.247 0.516 0.678 0.886 0.445 0.02 0.39 0.361 0.050
BMI: body mass index, WC: Waist circumference, FBG: Fasting blood glucose, HOMA-IR: Homeostasis model assessment for insulinresistance, HDL-C: high-density lipoprotein cholesterol, LDL-C: low-density lipoprotein cholesterol, TSH: thyroid stimulating hormone, FT4: Free T4, anti-TPO: anti-thyroid peroxidase, anti-Tg: antithyroglobulin.
Helicobacter pylori infection and autoimmune thyroid disease
DOI: 10.3109/07435800.2015.1015727
Table 2. Ultrasonographic characteristics of the H. pylori-positive and -negative patients.
Variable Heterogeneity
H. pylori () H. pylori (+) % (n) % (n)
Category Homogeneity Minimal Moderate Severe
36.7 24.5 34.7 4.1
(19) (12) (17) (2)
25 35 21.3 18.8
(21) (25) (17) (15)
Ultrasonograhy Normal Thyroiditis Thyroiditis with nodule Multinodular goiter Uninodular goiter
28.6 40.8 26.5 0 4.1
(14) (20) (13) (0) (2)
22.5 45 27.5 2.5 2.5
(18) (34) (21) (2) (2)
Nodule existence
73.5 (36) 26.5 (13)
Negative Positive
67.5 (54) 32.5 (25)
p
Table 4. Ultrasonographic characteristics of the H. pylori-positive patients according to the cagA status.
Variable
Category
cagA () % (n)
cagA (+) % (n)
p
Heterogeneity
Homogeneity Minimal heterogeneity Moderate heterogeneity Severe heterogeneity
26.5 35.3 17.6 20.6
(10) (12) (6) (7)
25.6 30.2 25.6 18.6
(11) (13) 40.05 (11) (8)
Ultrasonograhy
Normal Thyroiditis Thyroiditis with nodule Multinodular goiter Uninodular goiter
26.5 38.2 32.4 2.9 0
(10) (13) (11) (1) (0)
20.9 48.8 23.3 2.3 4.7
(9) 40.05 (21) (10) (1) (2)
Nodule existence
Negative Positive
61.8 (22) 38.2 (13)
0.023
0.726
0.474
3
72.1 (31) 40.05 27.9 (12)
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cagA: Cytokine-asscociated gene A.
Table 3. Demographics, biochemical characteristics, and thyroid volume in the H. pylori-positive patients, according to cagA status.
Male/female Age (years) BMI (kg m–2) WC (cm) FBG (mg dL–1) HDL-C (mg dL–1) LDL-C (mg dL–1) TG (mg dL–1) Fasting insulin (IU mL–1) HOMA-IR (%) TSH (mIU mL–1) FT4 (ng dL–1) Anti-TPO positivity (%) Anti-Tg positivity (%) Thyroid volume (mL) Nodule volume (mL)
cagA (+) (n ¼ 43)
cagA (–) (n ¼ 35)
p
15/28 39.3 ± 11.7 26.3 ± 4.5 89.1 ± 10.0 90.3 ± 12.8 45.6 ± 11.5 116 ± 30.3 121.5 ± 60.7 8.7 ± 4.3 1.9 ± 1.1 2.2 ± 2.1 0.98 ± 0.17 60 27.5 12.7 ± 8.1 1.1 ± 2.2
9/26 41.7 ± 11.8 29.9 ± 9.6 790.1 ± 9.5 88.1 ± 9.4 45.8 ± 12.5 103.1 ± 25.8 117.1 ± 38.9 8.8 ± 7.6 1.9 ± 1.6 1.8 ± 1.3 1.0 ± 0.2 58.5 23.5 11.8 ± 5.8 0.7 ± 2.1
40.05 40.05 40.05 40.05 40.05 40.05 0.05 40.05 40.05 40.05 40.05 40.05 40.05 40.05 40.05 40.05
BMI: body mass index, WC: Waist circumference, FBG: Fasting blood glucose, HOMA-IR: Homeostasis model assessment for insulinresistance, HDL-C: high-density lipoprotein cholesterol, LDL-C: low-density lipoprotein cholesterol, TSH: thyroid stimulating hormone, FT4: Free T4, anti-TPO: anti-thyroid peroxidase, anti-Tg: antithyroglobulin.
and the differences were significant (p ¼ 0.023; Table 2). Moreover, the H. pylori-positive patients were evaluated according to cagA status (Table 3); however, there was no significant difference in terms of HOMA-IR, metabolic parameters, ATD, or thyroid volume between the cagApositive and cagA-negative patients. There were no significant differences in ultrasonographic thyroid findings between the groups (Table 4). Finally, there was not any correlation between the cagA level, and thyroid total volume, nodule existence, thyroid autoantibody, or metabolic parameters, but there was a positive weak correlation between Hashimoto’s thyroiditis and H. pylori positivity (r ¼ 0.197, p ¼ 0.02).
Discussion We found frequency of autoimmune thyroid disease significantly higher in the H. pylori-positive group than in the
H. pylori-negative group. However, there was no significant difference in thyroid function tests or metabolic syndrome parameters between groups. Hashimoto’s thyroiditis is considered one of the most common autoimmune diseases as well as one of the most common endocrine disorders. Bassi et al. reported that H. pylori infection is correlated with autoimmune thyroid disease (8,9); however, other studies have not observed similar association (10,11). Autoimmune thyroid disease is characterized by the existence of thyroid autoantibodies against thyroid antigens, such as thyroid peroxidase antibodies and anti-thyroglobulin antibodies (20). It was reported that the development of ATD is multifactorial and that bacterial infections might play a pathogenic role. H. pylori is one the most commonly investigated infectious agents associated with thyroid disorders. Furthermore, cagA strains of H. pylori exhibit a nucleotide sequence similar to the thyroid peroxidase (TPO) sequence (21). Moreover, some studies reported that there is a strong correlation between H. pylori and thyroid autoantibodies, and that eradication of H. pylori infection is followed by a gradual decrease in the levels of thyroid autoantibodies (22). Another study reported that the levels of free T4 and free T3 were lower in H. pyloripositive individuals than H. pylori-negative individuals (23). It was also observed that cagA-positive strains of H. pylori were associated with an increase in the risk of ATD, especially in females (16), but data are inconsistent, as some studies reported that there is no difference in the serum levels of thyroid hormones or thyroid autoantibodies between H. pylori-positive and -negative individuals (10,11). As mentioned earlier, the impact of H. pylori infection on the thyroid, thyroid antibody positivity, Graves’ disease, and Hashimoto’s thyroiditis has been examined by many researchers; however, to the best of our knowledge, the present study is the first to include H. pylori-positive patients diagnosed histologically that underwent thyroid USG and thyroid antibody determination. In the present study, the participants were divided into two groups; the study group included H. pylori-positive patients (subgrouped according to cagA status) and an H. pylori-negative control group. In contrast to earlier studies, there wasn’t any significant difference between the two groups in terms of metabolic parameters, or IR, but there was a significant difference
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between the two groups in terms of positivity of thyroid autoimmunity and Hashimoto’s thyroiditis (12). The most important finding of this study is that patients with H. pylori infection could have long-term autoimmune thyroid disease; therefore, clinicians should be aware of ATD in such patients. It was reported that H. pylori infection leads to dyslipidemia, and an increment of total cholesterol, LDL-cholesterol, apolipoprotein B, lipoprotein A, and triglyceride levels, and a decrement of HDL-cholesterol levels might be observed (24). Furthermore, significantly higher levels of total cholesterol and LDL-cholesterol were noted in ischemic stroke patients with H. pylori infection compared to H. pylorinegative individuals (25,26). According to Elizalde et al., H. pylori infection did not significantly affect total cholesterol, triglycerides, or LDL cholesterol levels (27). In the present study, total cholesterol, triglycerides, LDL cholesterol, and HDL-cholesterol levels did not differ significantly between the study and control groups. On the other hand, several studies on H. pylori infection and its relationship to metabolic syndrome and IR reported inconsistent findings (28), which might have been due to differences in diagnostic methods. For instance, the presence of similar antigenic sites in anti-TPO and cagA could increase the rate of false positivity for H. pylori antibody titers (9). Additionally, H. pylori antibodies cannot discriminate between past and ongoing H. pylori infection. In conclusion, autoimmune thyroid disease must be considered in patients with H. pylori infection. However, cag A status has no effect on thyroid autoimmunity according to our study. Additional clinical trials are required to further explore the relationship between ATD and H. pylori infection.
Declaration of interest The authors declare that they have no conflicts of interest.
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9. Bassi V, Santinelli C, Iengo A, et al. Identification of a correlation between Helicobacter pylori infection and Graves’ disease. Helicobacter 2010;15:558–62. 10. Tomasi PA, Dore MP, Fanciulli G, et al. Is there anything to the reported association between Helicobacter pylori infection and autoimmune thyroiditis? Dig Dis Sci 2005;50:385–8. 11. Franceschi F, Satta MA, Mentella MC, et al. Helicobacter pylori infection in patients with Hashimoto’s thyroiditis. Helicobacter 2004;9:369. 12. Polyzos SA, Kountouras J, Zavos C, et al. The association between Helicobacter pylori infection and insulin resistance: a systematic review. Helicobacter 2011;16:79–88. 13. Manolakis AC, Tiaka EK, Kapsoritakis AN, et al. Increased fetuin A levels in Helicobacter pylori infection: a missing link between H. pylori and insulin resistance? Diabetologia 2010;54:472–4. 14. Pellicano R, Franceschi F, Saracco G, et al. Helicobacters and extragastric diseases. Helicobacter 2009;14:58–68. 15. Moran AP, Prendergast MM, Appelmelk BJ. Molecular mimicry of host structures by bacterial lipopolysaccharides and its contribution to disease. FEMS Immunol Med Microbiol 1996;16: 105–15. 16. Figura N, Di Cairano G, Lore` F, et al. The infection by Helicobacter pylori strains expressing CagA is highly prevalent in women with autoimmune thyroid disorders. J Physiol Pharmacol 1999;50:817–26. 17. Anil C, Akkurt A, Ayturk S, et al. Impaired glucose metabolism is a risk factor for increased thyroid volume and nodule prevalence in a mild to moderate iodine deficient area. Metabolism 2013;62: 970–5. 18. Erdogan M, Erdem N, Cetinkalp S, et al. Demographic, clinical, laboratory, ultrasonographic, and cytological features of patients with Hashimoto’s thyroiditis: results of a university hospital of 769 patients in Turkey. Endocrine 2009;36:486–90. 19. Matthews DR, Hosker JP, Rudenski AS, et al. Homeostasis Model Assessment: insulin resistance and beta-cell function from fasting blood glucose and insulin concentrations in man. Diabetologia 1985;28:412–19. 20. Rapoport B, McLachlan SM. Thyroid autoimmunity. J Clin Invest 2001;108:1253–9. 21. Tomb JF, White O, Kerlavage AR, et al. The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature. 1997; 388:539–47. 22. Bertalot G, Montresor G, Tampieri M, et al. Decrease in thyroid autoantibodies after eradication of Helicobacter pylori infection. Clin Endocrinol (Oxf) 2004;61:650–2. 23. Triantafillidis JK, Georgakopoulos D, Gikas A, et al. Relation between Helicobacter pylori infection, thyroid hormone levels and cardiovascular risk factors on blood donors. Hepatogastroenterology 2003;50(Suppl 2):cccxviii–xx. 24. Chimienti G, Russo F, Lamanuzzi BL, et al. Helicobacter pylori is associated with modified lipid profile: impact on Lipoprotein(a). Clin Biochem 2003;36:359–65. 25. Majka J, Ro´g T, Konturek PC, et al. Influence of chronic Helicobacter pylori infection on ischemic cerebral stroke risk factors. Med Sci Monit 2002;8:CR675–84. 26. Hoffmeister A, Rothenbacher D, Bode G, et al. Current infection with Helicobacter pylori, but not seropositivity to Chlamydia pneumoniae or cytomegalovirus, is associated with an atherogenic, modified lipid profile. Arterioscler Thromb Vasc Biol 2001;21: 427–32. 27. Elizalde JI, Pique´ JM, Moreno V, et al. Group of Study on Oesophageal and Diseases from The Spanish Gastroenterological Association. Influence of Helicobacter pylori infection and eradication on blood lipids and fibrinogen. Aliment Pharmacol Ther 2002;16:577–86. 28. Albaker WI. Helicobacter pylori infection and its relationship to metabolic syndrome: is it a myth or fact? Saudi J Gastroenterol 2011;17:165–9.
The relationship between cytotoxin-associated gene A positive Helicobacter pylori infection and autoimmune thyroid disease Muyesser Sayki Arslan1, Fuat Ekiz2, Murat Deveci2, Mustafa Sahin3, Oya Topaloglu1, Basak Karbek1, Esra Tutal1, Zeynep Ginis4, Erman Cakal1, Mustafa Ozbek1, Osman Yuksel2, and Tuncay Delibasi1 Department of Endocrinology and Metabolic Diseases and 2Department of Gastroenterology, Diskapi Yildirim Beyazit Training and Research Hospital, Ankara, Turkey, 3Department of Endocrinology and Metabolism, School of Medicine, Ankara University, Ankara, Turkey, and 4Department of Clinical Biochemistry, Diskapi Yildirim Beyazit Training and Research Hospital, Ankara, Turkey
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1
Abstract
Keywords
Objective: The aim of this study was to determine whether there is an association between cagA [cytotoxin-associated gene A] positivity and thyroid autoimmunity and thyroid volume. Methods: This prospective study included 78 Helicobacter pylori-positive (H. pylori) dyspeptic patients in the study group, and 50 age-, gender-, and body mass index-matched H. pylorinegative dyspeptic patients in the control group. All the controls were evaluated via upper gastrointestinal endoscopic biopsy or breath test, and were found as H. pylori negative. Gastric biopsy specimens were obtained via endoscopy and histological examination was performed for documentation of H. pylori. Results: In all, 55.1% (n ¼ 43) of the H. pylori-positive patients were cagA positive. There was no significant difference in metabolic syndrome parameters or thyroid function test results between the study and control groups. The frequency of anti-TPO and Hashimoto’s thyroiditis positivity was significantly higher in the study group than in the control group. Thyroid volume was higher and severe parenchymal heterogeneity was more common in the H. pylori-positive patients. Conclusions: H. pylori infection might be a risk factor for autoimmune thyroid disease and high thyroid volume in patients diagnosed with histological evaluation. However, cagA positivity has no additional effect on these parameters.
Helicobacter pylori, cytotoxin-associated gene A, thyroid function test
Introduction Helicobacter pylori (H. pylori) infection is the most common infectious disease and infects approximately 50% of the world’s population (1). Conditions characteristically associated with this infection are chronic gastritis, peptic ulcer disease, and gastric malignancies (2,3). When H. pylori infection is not adequately treated, it can cause extra-digestive disorders via local production and systemic diffusion of proinflammatory cytokines (4). Expression of the H. pylori protein cytotoxin-associated gene A (cagA) indicates the existence of a virulent H. pylori strain (5). It is reported that the presence of cagA protein is a risk factor for acne rosacea, stroke, and coronary heart diseases (6). Endocrine disorders thought to be associated with H. pylori infection include diabetes mellitus, autoimmune thyroid disease (ATD), dyslipidemia, obesity, primary hyperparathyroidism and osteoporosis (7). Several studies investigated the relationship between H. pylori infection and ATD, but the findings are
Correspondence: Muyesser Sayki Arslan, Department of Endocrinology and Metabolic Diseases, Diskapi Yildirim Beyazit Training and Research Hospital, Ankara 06110, Turkey. E-mail: [email protected]
History Received 16 June 2014 Revised 8 November 2014 Accepted 1 February 2015 Published online 30 March 2015
inconsistent. Bassi et al. investigated the correlation between the prevalence of H. pylori infection and ATD, and reported that H. pylori infection is correlated with Graves’ disease (8,9); however, other studies did not observe similar association (10,11). Current data shows that there is a potential association between insulin resistance (IR) and H. pylori infection; however, additional research is required to clarify the degree to which it affects IR (12). Although the pathophysiology of this relationship is not fully understood, an increase in the fetuin A level, and the release of vasoactive and proinflammatory substances are some of the main factors that contribute to IR (13,14). It is possible that H. pylori infection affects the thyroid via autoimmunity caused by molecular mimicry (15). It was reported that monoclonal antibodies to an H. pylori organism with cagA positivity reacted with follicular cells of the thyroid gland, and that an H. pylori strain possessing the cagA pathogenicity island has a gene encoding for an endogenous peroxidase (16). As ATD and IR are the most commonly diagnosed endocrine disorders, the present study was undertaken with the aim to determine if there is an association between cagA positivity, and Hashimoto’s thyroiditis and thyroid volume. We also evaluated metabolic parameters such as fasting glucose and IR that is known to affect thyroid volume (17).
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M. Sayki Arslan et al.
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Materials and methods This study was conducted at the Diskapi Yildirim Beyazit Training and Research Hospital in Ankara, Turkey. Participants were referred from the gastroenterology outpatient clinic to the endocrinology, diabetes and metabolism outpatient clinic. Seventy-eight patients in the study group were selected from those with gastrointestinal system symptoms evaluated by upper gastrointestinal (GI) endoscopy. All patients were histopathologically positive for H. pylori infection. None of the patients had any comorbidity, such as hypertension, diabetes, cardiovascular disorders, renal insufficiency, hepatic diseases, or thyroid dysfunction, and none were using any medications. The control group included 50 H. pylori-negative healthy volunteers. Helicobacter pylori negativity was determined by histopathological evaluation in 32 of the controls and by non-invasive C14 urea-breath test in 18 of the controls. Blood samples were collected from all of the participants and centrifuged. Serum was stored at 80 C in aliquots until the day of analysis. Serum H. pylori IgG antibodies to the cagA level were measured using a solidphase enzyme-linked immunosorbent assay (ELISA), according to the manufacturer’s instructions (DRG International, Inc., Springfield, NJ). Absorbance at 450 nm was measured using an ELISA reader (EPOCH). The ATD diagnosis was made to determine whether the patient had positivity of thyroid autoantibody (anti-Tg and anti-TPO) positivity and at least one of the criteria of thyroid parenchymal heterogeneity according to thyroid ultrasonography (USG), abnormality in thyroid function tests and diffuse goiter with physical examination (18). Following overnight fasting, venous blood samples for biochemical evaluation including routine renal and hepatic function, fasting glucose, insulin, high density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and triglyceride levels were taken from each participant. Thyroid function tests were assessed via measurement of FT4 and TSH, along with anti-thyroid peroxidase (anti-TPO) and anti-thyroglobulin (anti-Tg) using immunochemiluminescent assays (Immulite 2000; Diagnostic Products Corp., Los Angeles, CA) with the following normal ranges: TSH: 0.55–4.78 mIU/mL–1; FT4: 0.74– 1.52 ng/dL–1; anti-TPO: 0–57 IU/mL–1; anti-Tg: 564 IU/mL–1. IR was diagnosed based on the homeostasis model assessment (HOMA) index in all participants (19). The same physician measured each participant’s height, weight and waist circumference (WC). WC (in centimeters cm) was measured on a horizontal plane at the level of the umbilicus. The body mass index (BMI) was calculated by dividing body weight (kg) by height in square centimeters (cm2). Thyroid USG was performed in each participant by the same physician using a 12-MHz linear probe (Hitachi EUB 7000 HV, Tokyo, Japan). Thyroid gland volume was calculated according to the ellipsoid formula: volume (mL) ¼ length (cm) width (cm) depth (cm) /6 (9). Fine needle aspiration biopsy of the thyroid (FNAB) was performed in all thyroid nodules greater than 1 cm. Data were analyzed using SPSS v.18.0 for Windows (SPSS Inc., Chicago, IL). The normality of the distribution of continuous variables was determined using the
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Kolmogorov-Smirnov test. The Mann-Whitney test was used to compare variables that were not normally distributed and Student’s t test was used to compare variables with normal distribution. Categorical features and relationships between groups were assessed using an appropriate chi-square test. The level of statistical significance was set as p50.05.
Results Demographic, biochemical and thyroid volume details of the patients are shown in Table 1. As illustrated, the study included 128 volunteers (78 in the study group and 50 in the control group). The groups were matched for age, gender and BMI. The mean fasting blood glucose level and WC in both groups were similar. In addition, there wasn’t a significant difference in the mean LDL-C level or HOMA-IR between the two groups (Table 1). TSH levels were higher in the study group, but the difference was not significant. FT4 and FT3 levels were similar in both groups. The anti-TPO positivity rate was higher in the study group (p50.05), whereas the anti-Tg positivity rate was similar in both groups. Mean thyroid volume did not differ between the two groups. Moreover, nodule volume was higher in the study group than in the control group (p ¼ 0.05). We also evaluated the prevalence of positivity of ATD, and it was significantly higher in patients than in controls (70%, n ¼ 55 versus 30%, n ¼ 23; p ¼ 0.02, respectively). However, there was no significant difference in terms of HT prevalence according to cagA status. Fine needle thyroid aspiration biopsy was performed in all patients with risk factors and the results were reported as benign in all of them. The percentage of patients with thyroid nodules was similar in both groups (32.5 versus 26.5%; p ¼ 0.474). Mild, moderate, and severe thyroid heterogeneity was observed in 35, 21.3, and 18.8% of the study group, versus 24.5, 34.7 and 4.1% of the control group, respectively, Table 1. Demographics, biochemical characteristics, and thyroid volume in the H. pylori-positive and -negative patients.
Male/female Age (years) BMI (kg m2) WC (cm) FBG (mg dL1) HDL-C (mg dL1) LDL-C (mg dL1) TG (mg dL1) Fasting insulin (IU mL1) HOMA-IR (%) TSH (mIU/mL1) FT4 (ng dL1) Anti-TPO positivity (n, %) Anti-Tg positivity (n, %) Thyroid volume (mL) Nodule volume (mL)
H. pylori (+) (n ¼ 78)
H. pylori (–) (n ¼ 50)
p
24/54 40.9 ± 11.8 27.8 ± 7.5 89.6 ± 10.1 89.7 ± 11.6 45.5 ± 11.8 112.5 ± 31.7 118.4 ± 51.1 8.6 ± 5.8 1.9 ± 1.3 2.0 ± 1.7 1.0 ± 0.17 46 (58,2%) 22 (27,8%) 12.3 ± 7.0 0.9 ± 2.1
17/33 36.9 ± 11.8 25.9 ± 4.2 86.1 ± 8.2 86.5 ± 7.9 47.0 ± 10.7 111.0 ± 21.5 130.6 ± 65.4 9.2 ± 3.5 1.9 ± 0.8 1.9 ± 1.1 1.0 ± 0.3 33 (41,8) 12 (%24) 11.3 ± 4.1 0.11 ± 0.1
0.709 0.065 0.254 0.130 0.060 0.468 0.773 0.247 0.516 0.678 0.886 0.445 0.02 0.39 0.361 0.050
BMI: body mass index, WC: Waist circumference, FBG: Fasting blood glucose, HOMA-IR: Homeostasis model assessment for insulinresistance, HDL-C: high-density lipoprotein cholesterol, LDL-C: low-density lipoprotein cholesterol, TSH: thyroid stimulating hormone, FT4: Free T4, anti-TPO: anti-thyroid peroxidase, anti-Tg: antithyroglobulin.
Helicobacter pylori infection and autoimmune thyroid disease
DOI: 10.3109/07435800.2015.1015727
Table 2. Ultrasonographic characteristics of the H. pylori-positive and -negative patients.
Variable Heterogeneity
H. pylori () H. pylori (+) % (n) % (n)
Category Homogeneity Minimal Moderate Severe
36.7 24.5 34.7 4.1
(19) (12) (17) (2)
25 35 21.3 18.8
(21) (25) (17) (15)
Ultrasonograhy Normal Thyroiditis Thyroiditis with nodule Multinodular goiter Uninodular goiter
28.6 40.8 26.5 0 4.1
(14) (20) (13) (0) (2)
22.5 45 27.5 2.5 2.5
(18) (34) (21) (2) (2)
Nodule existence
73.5 (36) 26.5 (13)
Negative Positive
67.5 (54) 32.5 (25)
p
Table 4. Ultrasonographic characteristics of the H. pylori-positive patients according to the cagA status.
Variable
Category
cagA () % (n)
cagA (+) % (n)
p
Heterogeneity
Homogeneity Minimal heterogeneity Moderate heterogeneity Severe heterogeneity
26.5 35.3 17.6 20.6
(10) (12) (6) (7)
25.6 30.2 25.6 18.6
(11) (13) 40.05 (11) (8)
Ultrasonograhy
Normal Thyroiditis Thyroiditis with nodule Multinodular goiter Uninodular goiter
26.5 38.2 32.4 2.9 0
(10) (13) (11) (1) (0)
20.9 48.8 23.3 2.3 4.7
(9) 40.05 (21) (10) (1) (2)
Nodule existence
Negative Positive
61.8 (22) 38.2 (13)
0.023
0.726
0.474
3
72.1 (31) 40.05 27.9 (12)
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cagA: Cytokine-asscociated gene A.
Table 3. Demographics, biochemical characteristics, and thyroid volume in the H. pylori-positive patients, according to cagA status.
Male/female Age (years) BMI (kg m–2) WC (cm) FBG (mg dL–1) HDL-C (mg dL–1) LDL-C (mg dL–1) TG (mg dL–1) Fasting insulin (IU mL–1) HOMA-IR (%) TSH (mIU mL–1) FT4 (ng dL–1) Anti-TPO positivity (%) Anti-Tg positivity (%) Thyroid volume (mL) Nodule volume (mL)
cagA (+) (n ¼ 43)
cagA (–) (n ¼ 35)
p
15/28 39.3 ± 11.7 26.3 ± 4.5 89.1 ± 10.0 90.3 ± 12.8 45.6 ± 11.5 116 ± 30.3 121.5 ± 60.7 8.7 ± 4.3 1.9 ± 1.1 2.2 ± 2.1 0.98 ± 0.17 60 27.5 12.7 ± 8.1 1.1 ± 2.2
9/26 41.7 ± 11.8 29.9 ± 9.6 790.1 ± 9.5 88.1 ± 9.4 45.8 ± 12.5 103.1 ± 25.8 117.1 ± 38.9 8.8 ± 7.6 1.9 ± 1.6 1.8 ± 1.3 1.0 ± 0.2 58.5 23.5 11.8 ± 5.8 0.7 ± 2.1
40.05 40.05 40.05 40.05 40.05 40.05 0.05 40.05 40.05 40.05 40.05 40.05 40.05 40.05 40.05 40.05
BMI: body mass index, WC: Waist circumference, FBG: Fasting blood glucose, HOMA-IR: Homeostasis model assessment for insulinresistance, HDL-C: high-density lipoprotein cholesterol, LDL-C: low-density lipoprotein cholesterol, TSH: thyroid stimulating hormone, FT4: Free T4, anti-TPO: anti-thyroid peroxidase, anti-Tg: antithyroglobulin.
and the differences were significant (p ¼ 0.023; Table 2). Moreover, the H. pylori-positive patients were evaluated according to cagA status (Table 3); however, there was no significant difference in terms of HOMA-IR, metabolic parameters, ATD, or thyroid volume between the cagApositive and cagA-negative patients. There were no significant differences in ultrasonographic thyroid findings between the groups (Table 4). Finally, there was not any correlation between the cagA level, and thyroid total volume, nodule existence, thyroid autoantibody, or metabolic parameters, but there was a positive weak correlation between Hashimoto’s thyroiditis and H. pylori positivity (r ¼ 0.197, p ¼ 0.02).
Discussion We found frequency of autoimmune thyroid disease significantly higher in the H. pylori-positive group than in the
H. pylori-negative group. However, there was no significant difference in thyroid function tests or metabolic syndrome parameters between groups. Hashimoto’s thyroiditis is considered one of the most common autoimmune diseases as well as one of the most common endocrine disorders. Bassi et al. reported that H. pylori infection is correlated with autoimmune thyroid disease (8,9); however, other studies have not observed similar association (10,11). Autoimmune thyroid disease is characterized by the existence of thyroid autoantibodies against thyroid antigens, such as thyroid peroxidase antibodies and anti-thyroglobulin antibodies (20). It was reported that the development of ATD is multifactorial and that bacterial infections might play a pathogenic role. H. pylori is one the most commonly investigated infectious agents associated with thyroid disorders. Furthermore, cagA strains of H. pylori exhibit a nucleotide sequence similar to the thyroid peroxidase (TPO) sequence (21). Moreover, some studies reported that there is a strong correlation between H. pylori and thyroid autoantibodies, and that eradication of H. pylori infection is followed by a gradual decrease in the levels of thyroid autoantibodies (22). Another study reported that the levels of free T4 and free T3 were lower in H. pyloripositive individuals than H. pylori-negative individuals (23). It was also observed that cagA-positive strains of H. pylori were associated with an increase in the risk of ATD, especially in females (16), but data are inconsistent, as some studies reported that there is no difference in the serum levels of thyroid hormones or thyroid autoantibodies between H. pylori-positive and -negative individuals (10,11). As mentioned earlier, the impact of H. pylori infection on the thyroid, thyroid antibody positivity, Graves’ disease, and Hashimoto’s thyroiditis has been examined by many researchers; however, to the best of our knowledge, the present study is the first to include H. pylori-positive patients diagnosed histologically that underwent thyroid USG and thyroid antibody determination. In the present study, the participants were divided into two groups; the study group included H. pylori-positive patients (subgrouped according to cagA status) and an H. pylori-negative control group. In contrast to earlier studies, there wasn’t any significant difference between the two groups in terms of metabolic parameters, or IR, but there was a significant difference
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4
M. Sayki Arslan et al.
between the two groups in terms of positivity of thyroid autoimmunity and Hashimoto’s thyroiditis (12). The most important finding of this study is that patients with H. pylori infection could have long-term autoimmune thyroid disease; therefore, clinicians should be aware of ATD in such patients. It was reported that H. pylori infection leads to dyslipidemia, and an increment of total cholesterol, LDL-cholesterol, apolipoprotein B, lipoprotein A, and triglyceride levels, and a decrement of HDL-cholesterol levels might be observed (24). Furthermore, significantly higher levels of total cholesterol and LDL-cholesterol were noted in ischemic stroke patients with H. pylori infection compared to H. pylorinegative individuals (25,26). According to Elizalde et al., H. pylori infection did not significantly affect total cholesterol, triglycerides, or LDL cholesterol levels (27). In the present study, total cholesterol, triglycerides, LDL cholesterol, and HDL-cholesterol levels did not differ significantly between the study and control groups. On the other hand, several studies on H. pylori infection and its relationship to metabolic syndrome and IR reported inconsistent findings (28), which might have been due to differences in diagnostic methods. For instance, the presence of similar antigenic sites in anti-TPO and cagA could increase the rate of false positivity for H. pylori antibody titers (9). Additionally, H. pylori antibodies cannot discriminate between past and ongoing H. pylori infection. In conclusion, autoimmune thyroid disease must be considered in patients with H. pylori infection. However, cag A status has no effect on thyroid autoimmunity according to our study. Additional clinical trials are required to further explore the relationship between ATD and H. pylori infection.
Declaration of interest The authors declare that they have no conflicts of interest.
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