|Year : 2019 | Volume
| Issue : 3 | Page : 251-258
Association between Helicobacter pylori infection and autoimmune Hashimoto’s thyroiditis
Naglaa Gamal AboElazaim1, Ahmed AbdElrahman Tahoun1, Magdy AbdElkarim Al Matboly2, Ahmad Fathy AbdElaziz1, Rafik Abdelatef Metwally1
1 Department of Clinical Pathology, Faculty of Medicine, Al-Azhar University, Cairo, Egypt
2 Department of Internal Medicine, Faculty of Medicine, Al-Azhar University, Cairo, Egypt
|Date of Submission||17-Mar-2019|
|Date of Decision||20-Mar-2019|
|Date of Acceptance||10-Apr-2019|
|Date of Web Publication||26-Nov-2019|
Rafik Abdelatef Metwally
Department of Clinical Pathology, Faculty of Medicine, Al-Azhar University, Cairo, 11884
Source of Support: None, Conflict of Interest: None
Background Autoimmune thyroid diseases are multifactorial conditions that result from genetic predisposition in combination with environmental risk factors. Some bacteria and viruses are suspected of being able to mimic the antigenic profile on the thyroid membrane and play an important role in the onset of autoimmune diseases.
Aim The aim of this study was directed to investigate the association between Helicobacter pylori infection and Hashimoto’s thyroiditis (HT).
Patients and methods The present study included 100 patients divided into two groups: patient group included 70 patients, newly diagnosed as having HT and control group included 30 age-matched and sex-matched apparently healthy individuals. All participants were subjected to assay of serum thyroid-stimulating hormone, free T3, free T4, thyroperoxidase antibodies, thyroglobulin antibodies, serum H. pylori Ab IgG, and fecal H. pylori antigen.
Results In the present work, there was abnormal elevation of thyroid-stimulating hormone in 60 (85.7%) patients, compared with just one (3.3%) patient of control group, with statistically significant difference between groups. In the present work, there was significant and proportional (positive) correlation between H. pylori IgG and each of thyroperoxidase and anti-thyroglobulin values.
Conclusion There is a significant association between HT and H. pylori infection. This association might be related to the disease severity and might reflect the need of adding anti-H. pylori medications as additional choice in management of HT.
Keywords: Hashimoto’s thyroiditis, Helicobacter pylori, H. pylori IgG, thyroperoxidase and anti-thyroglobulin
|How to cite this article:|
AboElazaim NG, Tahoun AA, Al Matboly MA, AbdElaziz AF, Metwally RA. Association between Helicobacter pylori infection and autoimmune Hashimoto’s thyroiditis. Al-Azhar Assiut Med J 2019;17:251-8
|How to cite this URL:|
AboElazaim NG, Tahoun AA, Al Matboly MA, AbdElaziz AF, Metwally RA. Association between Helicobacter pylori infection and autoimmune Hashimoto’s thyroiditis. Al-Azhar Assiut Med J [serial online] 2019 [cited 2020 Jul 14];17:251-8. Available from: http://www.azmj.eg.net/text.asp?2019/17/3/251/271681
| Introduction|| |
Helicobacter pylori is microaerophilic, spiral, and gram-negative bacteria. Overall, 90% of duodenal ulcer and approximately 76–95% of gastric cancers are associated with H. pylori infection .
H. pylori infection is nearly 50–75% of the worldwide population; in Egypt, H. pylori prevalence is 72.38% [studied in a cohort study by the Egyptian ministry of health and the US Naval Medical Research Unit (namru-3)]. This high prevalence is owing to low socioeconomic status and living in rural areas . In the developing countries 70% of people are affected, whereas approximately 25–50% in the developed countries . In the United States, the annual cost concerning peptic ulcer disease is estimated to be $6 billion, and gastric cancer kills more 700 000 people per year in cosmopolitan areas .
Chronic autoimmune (Hashimoto’s) thyroiditis is the most common and largely studied organ-specific autoimmune disorder in humans. The prevalence rate of Hashimoto’s thyroiditis (HT) is 1–4% with incidence of 30–60/100 000 population per year. It is the most common cause of hypothyroidism in iodine-replete areas worldwide. The diagnosis of the disease is based on positive serum thyroid autoantibodies mainly thyroperoxidase antibodies (TPOAbs), thyroglobulin antibodies (TgAbs), and the characteristic echo graphic pattern of diffuse or irregular hypoechogenicity of the thyroid gland .
H. pylori infection association with extragastric diseases such as immune thrombocytopenic purpura, iron-deficiency anemia, and coronary heart disease is widely approved. However, there is significant decrease of free T3 and free T4 in H. pylori infection . The risk factors for autoimmune thyroid diseases (AITDs) remain largely unknown, and whether H. pylori infection is associated with AITDs is still controversial .
| Aim of the work|| |
The aim of this study was directed to investigate the association between H. pylori infection and HT.
| Patients and methods|| |
This case–control study was conducted at the Internal Medicine Outpatient Clinics at AL-Hussein University Hospital over the period between November 2016 and January 2019. The study was approved by the Ethics Board of Al-Azhar University.
This study included 70 patients who were newly diagnosed as having HT guided by clinical diagnosis and detection of thyroid-stimulating hormone (TSH), free T3, free T4, TPOAbs, and TgAbs. There were 17 males and 53 females, with their age ranging between 19 and 63 years.
The following were the inclusion criteria:
- Newly diagnosed patients with HT.
- Patients not receiving specific medications for thyroid illness.
The following were the exclusion criteria:
- Patients received medication for HT or H. pylori infection before the study.
- Patients with chronic systemic diseases such as chronic renal failure, chronic liver disease, and diabetes mellitus.
Thirty age-matched and sex-matched apparently healthy individuals with normal thyroid profile (TSH, free T3, and free T4) were included as a control group. There were 11 males and 19 females with their age ranging between 20 and 58 years.
All patients included in this study were subjected to the following:
- Full clinical assessment including thorough medical history and clinical examination.
- Laboratory assessment as follows:
- Routine investigations: CBS, RBS, ALT, AST, bilirubin, serum creatinine, and blood urea.
- Thyroid profile: TSH, free T3, and free T4.
- TPOAbs and TgAbs.
- Serum quantitative H. pylori IgG.
- Detection of H. pylori antigen in stool samples by rapid test.
- Confirmation of H. pylori antigen in stool positive samples by PCR.
Venous blood samples and stool samples were obtained from all participants (patients and controls) after taking written medical consent for participation in the study.
From each participant in the study, 7 ml venous blood sample was withdrawn, and divided as follow:
- 1 ml blood on EDTA for complete blood count (CBC).
- 6 ml of blood was left at room temperature for 20 min to be clotted and then centrifuged at 3000 rpm for 10 min, and the separated serum was divided into three aliquots: one used for routine laboratory investigations; the second used for immediate assay of TSH, FT3, FT4, TPOAbs, and TgAbs; and the third aliquot was kept frozen at −20°C until used for assay of H. pylori IgG by enzyme-linked immunosorbent assay (ELISA).
Stool samples were obtained and examined by one-step test for the detection of H. pylori Ag on the same day of collection. Positive samples were kept frozen at −20°C for confirmation by molecular diagnosis (PCR).
- CBC was done on fully automated cell counter Sysmex KX- 21NTM (Cobas e411 Analyzer, Roche Diagnostics, Indianapolis, Indiana, USA).
- Renal function test, liver function tests, and RBS were done by Cobas c 311 fully automated chemistry analyzer (Roche Diagnostics).
- Detection of TSH, free T3, and free T4, anti-Tg and anti-TPO was done.
- Detection of H. pylori IgG in serum by quantitative (EIA) enzyme immunoassay test kit (Catalog Number: 10207; Chemux Bioscience Inc., South San Francisco, California, USA).
- Detection of H. pylori Ag in stool sample by one-step test using the ABON Diagnostics Inc. (Woodland Hills, California, USA).
- Detection of H. pylori Ag in stool sample by PCR run on the Bio-Rad CFX96 TouchTM Real-Time PCR Detection System.
DNA extraction protocol was followed according to PSP spin stool DNA kit (Invitek, Germany) which is optimized for DNA extraction from stool samples.
The rapid thermal annealing (RTA)-spin filter was transmitted to a new RTA receiver tube, and 500 μl of wash buffer one was pipetted onto the RTA-spin filter, centrifuged at 10.000 rpm for 1 min, then the flow-through and the RTA receiver tube were discarded.
The RTA-spin filter was then placed in a new RTA receiver tube, and 700 μl of wash buffer two pipetted onto the RTA-spin filter and centrifuged at 10.000 rpm for 1 min, and then the flow-through was discarded (the RTA receiver tube was centrifuged again for 3 min at maximum speed to eliminate any traces of ethanol then discarded).
The RTA-spin filter was transmitted to a new receiver tube of 1.5 ml, and 100–200 μl of elution buffer D preheated to 70°C was pipetted directly onto the center of the membrane of the RTA-spin filter and incubated for 1 min at room temperature and then centrifuged at 10.000 rpm for 1 min.
The eluted DNA was stored immediately at −20°C, and the RTA-spin filter was discarded.
Determination of DNA yield, concentration, and purity:
- Determination of DNA yields from DNA eluate measured was done by reading absorbance at 260 nm.
- Determination of DNA purity was done by calculating the reading ratio of absorbance at 260 nm to absorbance at 280 nm. Pure DNA had ratio of 1.7–1.9.
It was run on the Bio-Rad CFX96 TouchTM Real-Time PCR Detection System.
The following materials were used:
- DNA template.
- DreamTaq Green PCR master mix, which was supplied by Fermentas (Waltham, Massachusetts, USA) and composed of DreamTaq DNA polymerase, optimized DreamTaq Green buffer, MgCl2, and dNTPs.
- Two primers were supplied as lyophilized powder to be reconstituted upon use.
- The 16 s rRNA gene (399 bp).
- Heli f: 5′-AAC GAT GAA GCT TCT AGC TTG CTA G3′.
- Helir: 5′GTG CTT ATT CAT CAG ATA CCG TCA T3′.
- The glmM gene (294 bp).
- F: 5-GGATAAGCTTTTAGGGGTGTTAGGGG-3.
- R: 5-GCTTACTTTCTAACACTAACGCGC-3.
- Nuclease-free water.
- Negative control: injection water (sterile nuclease-free water).
- Positive control: Escherichiacoli VC167.
The 16 s r RNA
Initial denaturation was done at 94°C for 5 min, then 35 cycles of 94°C for 30 s, 56°C for 1 min, and 72°C for 1 min, followed by a final extension of 72°C for 10 min with initial delay for 1 min. amplified products were stored at −20°C.
The glmM gene (294 bp)
Initial denaturation was done at 94°C for 5 min for one cycle, then 35 cycles at 94°C for 1 min, annealing at 56°C for 1 min and elongation at 72°C for 2 min, and the final elongation step by one cycle at 72°C for 7 min. Amplified products were stored at −20°C.
Sensitivity of the primers was determined by testing other bacterial strains from related genus, e.g. E. coli.
Detection of the amplification products
Agarose gel electrophoresis is based on charge, size, and shape of the molecules used to separate molecules.
- The gel is examined under ultraviolet light, where the ethidium bromide intercalates between the bases of the DNA, which fluoresces. Photographs were taken using a Polaroid camera which is connected to computer system.
- Different bands ranging from 100 to 2000 bp are specified by a marker.
- To exclude any source of contamination, the negative control is examined.
- Positive control is compared with samples.
- Positive samples gave band at 450 bp position.
Data were statistically described in terms of mean±SD, median and range, or frequencies (number of cases) and percentages when appropriate (skewed data). Comparison of numerical variables between the study groups was done using Student’s t test for independent samples. For comparing categorical data, χ2 test was performed. Exact test was used instead when the expected frequency is less than 5. P values less than 0.05 was considered statistically significant. The diagnostic performance of the different parameters was evaluated using receiver operating characteristics curve analysis. All statistical calculations were done using computer programs SPSS (Statistical Package for the Social Science; SPSS Inc., Chicago, Illinois, USA) version 15, for Microsoft Windows.
| Results|| |
The present study included 100 patients divided into two groups: patient group (70 patients) and control group (30 normal patients) ([Table 1]).
In the present study, the patient age ranged from 19 to 63 years, and there was no significant difference between study and control groups (36.70±9.73 vs. 37.96±10.93 years, respectively) ([Table 2]).
In the present study, females represented 72% of all studied populations, and there was no significant difference between study and control groups, as females represented 75.7% of study group compared with 63.3% of the control group ([Table 3]).
|Table 3 Normal and abnormally elevated thyroid-stimulating hormone in studied groups|
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In study group, there was abnormal elevation of TSH in 60 (85.7%) patients, compared with just one (3.3%) patient of control group, with a statistically significant difference between groups ([Table 4]).
In the present work, there were 10% of patients in study group who had decreased values of free T3 and 11.4% had increased levels of free T3. This was compared with 0.0 and 23.3%, respectively, in the control group. There was significant difference between groups ([Table 5]).
Decreased values of free T4 were reported in 28.6% of study group compared with none in control group, whereas increased values of free T4 was reported in 7.1% in the study compared with 3.3% of the control group, with significant difference between both groups ([Table 6]).
In the present work, there was a statistically significant increase of abnormally increased values of TPO in the study when compared with the control group (77.1 vs. 0.0%, respectively) ([Table 7]).
In the present study, there was a statistically significant increase of abnormally increased values of anti-Tg in study when compared with the control group (82.9 vs. 0.0%, respectively) ([Table 8]).
|Table 8 Results of Helicobacter pylori antigen in stool among studied populations|
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Regarding H. pylori antigen in the stool, it was positive in 38 (54.3%) patients in the study group, compared with six (20%) patients of the control group, and there was significant increase in study when compared with control groups ([Table 9]).
|Table 9 Correlation between Helicobacter pylori IgG and other studied variables in study group|
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In the present work, there was mild, significant, and proportional (positive) correlation between H. pylori IgG and each of TPO and anti-Tg values. Otherwise, no other significant correlation was found ([Table 10], [Figure 1]).
|Table 10 The suggested cutoff values for thyroid-stimulating hormone, free T3, free T4, thyroperoxidase, anti-thyroglobulin, and Helicobacter pylori IgG levels between patient and control groups|
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|Figure 1 Multi-ROC curve analysis showing the diagnostic performance of TSH, free T3, free T4, TPO, anti-Tg, and Helicobacter pylori IgG for discriminating of patients with Hashimoto disease from healthy controls. ROC, receiver operating characteristics; Tg, thyroglobulin; TPO, thyroperoxidase; TSH, thyroid-stimulating hormone.|
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In the present study, the suggestive cutoff values for diagnosis of Hashimoto’s disease are 3.43, 3.37, 1.03, 2.76, 3.01, and 12.5; sensitivity values are 88.6, 54.3, 82.0, 80.0, 88.6, and 68.6%; and specificity values are 90.0, 67.0, 82.0, 80.0, 96.7, and 54.00% for TSH, FT3, FT4, TPO, anti-Tg, and H. pylori IgG, respectively.
| Discussion|| |
H. pylori is progressively being associated with extradigestive diseases. H. pylori is vastly accepted as a cause of iron-deficiency anemia and idiopathic thrombocytopenia, but the agreement is still not out on other diseases .
The most widespread autoimmune endocrine disorder and the most common cause of hypothyroidism is considered HT. The prevalence rate of HT is 1–4% with incidence of 30–60/100 000 population per year. The classic form of HT is associated with hypothyroidism, euthyroidism, or occasionally hyperthyroidism .
The present work aimed to investigate the association between H. pylori infection and HT.
Our results showed that the age of patients in the study group ranged from 19 to 63 years, which suggests that AITD mostly occurs in adult age. This fact is also confirmed by Akamizu et al.  who claim that thyroid diseases occur specially from 20 to 55 years during the middle decades but may be seen in any age, even in children.
In the present study, females represented 75.7% of study group, which is nearly the same results by Hamid , where 82% of patients with HT were females because sex hormones may play a role in this sex, as reported in several previous studies. Sex hormones, estrogen, progesterone, and testosterone are considered to mediate the immune response to many of the sex-based autoimmune diseases . The sex hormone directly interacts with immune system via the receptor on or inside immune cells. Steroid hormones are known by their effect in antibody production and proliferation of immune cells .
In the present work, there was no significant difference between study and control groups regarding CBC findings and liver and renal function tests.
In the study group, there was abnormal elevation of TSH in 60 (85.7%) patients, compared with just one (3.3%) patient of control group, with a statistically significant difference between both groups. Regarding the level of free T3, 10% of patients in the study group had decreased values of free T3 and 11.4% had increased levels of free T3. This was compared with 0.0 and 23.3%, respectively, in the control group, with a significant difference between both groups. Meanwhile values of free T4 were found to be decreased in 28.6% of study group compared with none in the control group, whereas increased values of free T4 were reported in 7.1% in the study compared with 3.3% of the control group, with a significant difference between both groups.
Thyroid TPOAbs and anti-TgAbs possess its strong value in the diagnosis of thyroid autoimmunity. There was a statistically significant increase of abnormally increased values of TPO in the study when compared with the control group (77.1 vs. 0.0%, respectively). There was a statistically significant increase of abnormally increased values of anti-Tg in study when compared with the control group (82.9 vs. 0.0%, respectively).
In the present work, when comparing positive and negative H. pylori-infected patients as detected by PCR, there was a significant increase of TPO, anti-Tg, and H. pylori IgG in positive when compared with negative patients. Moreover, there was a significant positive correlation between serum H. pylori IgG and each of TPO and anti-Tg values.
In the current study, the suggestive cutoff values for diagnosis of Hashimoto’s disease are 3.43, 3.37, 1.03, 2.76, 3.01, and 12.5; sensitivity values are 88.6, 54.3, 82.0, 80.0, 88.6, and 68.6%; and specificity values are (90.0, 67.0, 82.0, 80.0, 96.7, and 54.00% for TSH, FT3, FT4, TPO, anti-Tg, and H. pylori IgG, respectively, and there is a linear correlation from receiver operating characteristics curve between TSH, FT3, FT4, TPO, anti-Tg, and H. pylori IgG.
Our results showed significant association between HT and H. pylori infection. This association supports the hypothesis that H. pylori acts as a risk factor for HT and may be related to the disease initiation and severity and may reflect the need of adding anti-H. pylori medications in management of HT.
The current study could not explain the mechanism by which H. pylori can trigger HT. Bogdanos and Vergani  agree with the result of the present study in that the exact mechanisms by which exposure to a microbe induce more than one autoimmune manifestation are not well defined but cross-reactive responses against a microbe and self-antigens have been documented.
Our results are consistent with most of previous studies such as results showed by Hou et al. . Their study presented a systematic meta-analysis regarding the correlation between H. pylori infection and AITDs. Fifteen articles including 3046 cases were selected, including 1716 observational and 1330 control cases. They found H. pylori infection was positively correlated with the occurrence of AITD odds ratio (OR)=2.25, 95% confidence interval (CI): 1.72–2.93. Infections with H. pylori strains positive for CagA were positively correlated with AITD (OR=1.99, 95% CI: 1.07–3.70). There was no significant difference between infections examined using ELISA and other methods. Patients of Grave’s disease and HT were more susceptible to infection of H. pylori (Grave’s disease: OR=2.78, 95% CI: 1.68–4.61; HT: OR=2.16, 95% CI: 1.44–3.23), whereas the rate of infection of H. pylori did not differ between Grave’s disease and HT (χ2=3.113, P=0.078). They concluded that infection of H. pylori correlated with Grave’s disease and HT, and the eradication of H. pylori infection might be reduce thyroid autoantibodies.
In accordance with our results is the study done by Zainab  where she studied the association of H. pylori infection with HT, in a case–control study that involved patients with HT and control group (hyperthyroidism and healthy persons in equal numbers). The study groups were investigated regarding H. pylori IgG, anti-TPOAb and anti-TgAb using ELISA technique; in addition, demographic data were taken from the study groups. Her results showed that the strength and direction of the relationship between the concentrations of H. pylori IgG and TPOAbs in HT group revealed positive correlation (r=0.6241). She concluded the infection with H. pylori is one of predisposing factors to HT.
In contrast to our results, Shmuely et al.  investigated women (in which HT is more common), H. pylori IgG (using ELISA technique), CagA antigen (Western blot assay), TPOAbs and TgAbs, and found no association between H. pylori infection and HT in women. Moreover, they stated that family history of thyroid dysfunction is considered a risk factor for HT. This disagreement with our study may be owing to the differences in the sex type of cases and control; on the contrary, there is a considerable geographic variation in the risk of thyroid malfunction between different region. Moreover, the prevalence of the bacterium is affected by geographical distribution and socioeconomic status.
| Conclusion|| |
Our results showed significant association between HT and H. pylori infection. This association supports the hypothesis that H. pylori acts as a risk factor for HT and may be related to the disease initiation and severity, which reflects the need of adding anti-H. pylori medications in management of HT.
- It is advised that patients with H. pylori infection should be followed up regularly by repeated testing of TgAb and TPOAb in their sera, for earlier detection of subclinical autoimmune thyroid dysfunction before any changes in TSH values occur.
- It is recommended also to consider that patients with autoimmune thyroid dysfunction should be examined for detection of H. pylori infection even without dyspeptic symptoms because there is positive correlation between H. pylori infection and AITD, especially those on high doses of thyroxin.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Ramesh R, Sheng LW, Jing L, Ying XW, Qian WR, Chang QY. Helicobacter pylori
infection: a recent approach to diagnosis and management. J Biomed 2017; 2:45–56.
Mohammad MA, Hussein L, Coward A, Jackson SJ. Prevalence of Helicobacter pylori
infection among Egyptian children: impact of social background and effect on growth. Public Health Nutr 2008; 11:230–236.
Safavi M, Sabourian R, Foroumadi A. Treatment of Helicobacter pylori
infection: current and future insights. World J Clin Cases 2016; 4:5–19.
Wagner AP. Vibratory stimulation of thyroid epithelial mimics hormonal stimulation. Iowa Res Online 2017; 56:72.
Korani M, Elshayeb E, Sonbal A. Helicobacter pylori
infection: association with Hashimoto’s thyroiditis. Gastroenterol Hepatol 2016; 4: 11–11.
Yun MC, Tae YK, Eui YK, Eun KJ, Min JJ, Won GK, Young KS, Won BK. Helicobacter pylori
association between thyroid autoimmunity and Helicobacter pylori
infection. Korean J Intern Med 2017; 32:309–313.
Testerman TL, Morris J. Beyond the stomach: an updated view of Helicobacter pylori
pathogenesis, diagnosis, and treatment. World J Gastroenterol 2014; 20:12781–12808.
Shmuely H, Melzer E, Braverman M, Domniz N, Yahav J. Helicobacter pylori
infection is associated with advanced colorectal neoplasia. Scand J Gastroenterol 2014; 49:35–42.
Akamizu T, Amino N, DeGroot LJ. Hashimoto’s thyroiditis. Endotext 2013; 20:12–15.
Hamid ZA. The possible role of Helicobacter pylori
infection in hashimoto’s thyroiditis. Fac Med Baghdad 2017; 59:79–82.
Zandman-Goddard G, Peeva E, Shoenfeld Y. Gender and autoimmunity. Autoimmunity Rev 2007; 6:366–372.
Fairweather D, Frisancho-Kiss S, Rose N. Differences in autoimmune disease from a pathological perspective. Am J Pathol 2008; 173:600–609.
Bogdanos D, Vergani D. Origin of cross-19 reactive autoimmunity in primary biliary cirrhosis. Liver Int J 2006; 26:633–635.
Hou Y, Sun W, Zhang C, Wang T, Guo X, Wu L, Qin L, Liu T. Meta-analysis of the correlation between Helicobacter pylori
infection and autoimmune thyroid diseases. Oncotarget 2017; 8:115691–115700.
Shmuely H, Shimon I, Gitter LA. Helicobacter pylori
infection in women with Hashimoto thyroiditis: a case-control study. Medicine (Baltimore) 2016; 95:40–74.
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8], [Table 9], [Table 10]