H.pylori belongs to the family Helicobacteraceae. It is a transmissible and pathogenic gram-negative spiralshaped bacterium thought to be a contaminant of digested food as opposed to being a true colonizer of the gastric mucosa ,they intentionally ingested the bacterium and subsequently developed persistent gastritis, Later, it was found that the bacteria are strongly associated with multiple upper gastrointestinal disorders, such as chronic gastritis, peptic ulcer disease, gastric mucosa-associated lymphoid tissue (MALT) lymphoma, and gastric cancer.[1]

H.pylori strains have different genes about 1600 genes encoding virulence factors (encoded proteins) which are secreted membrane-associated or translocated into cytosol of the host cells via the IV type secretion system, where they can affect the host cell functions. The most studied virulence factors implicated in the pathogenicity of H.pylori are produced by strains have the following genes:  cytotoxin-associated gene A (cagA), duodenal ulcer (DU) promoting gene (dupA), vacuolating cytotoxin gene (vacA), induced by contact with epithelium gene (iceA), blood group antigen-binding adhesin 

(babA), sialic acid binding adhesin (sabA), outer inflammatory protein A (oipA), adherence-associated lipoprotein A and B (alpA/B) [2,3]

 The researcher [4]also mentioned that H.pylori outer membrane proteinQ (hopQ), gamma-glutamyl transpeptidase (GGT) and high-temperature requiring protein (HtrA) and the  regulation and function of the proteins are complex processes.

Vitamin D3, also called calciferol, is one of the four fat soluble (dissolve in fat) Vitamins (A, D, E, and K) stored in body tissues. Vitamin D3 is the only Vitamin that can be synthesized by the human body,human body can produce Vitamin D3 in the skin when exposed to sunlight, namely the ultraviolet B radiation,other sources of Vitamin D3 include dietary supplements and food such as fortified milk, fortified cereals, fatty fish, cod-liver oil, mushrooms, and egg yolks, Vitamin D3 is initially present in an inactivated form, either as ergocalciferol or as cholecalciferol,the synthesis of the active form of Vitamin D, calcitriol,requires two hydroxylations, which take place in the liver and kidney, Calcitriol binds to Vitamin D receptors (VDRs), which are found throughout the entire human body and present numerous binding sites , Vitamin D3 regulates multiple biologic processes[5]

The researcher [6] noted the Vitamin D3 deficiency is associated with numerous aging-related diseases, such as hypertension, type 2 diabetes mellitus, and coronary heart diseases,in addition,The metabolism of calcium, phosphate, and bone is controlled by Vitamin D,also,numerous studies reported that the majority of students (94%) believed that bone and skeletal disorders could be caused by Vitamin D3 deficiency, Also, deficiency of Vitamin D3 is linked to high incidences of  autoimmune disorders, cardiovascular disorders, psychological disorders, and cancer, Vitamin D3 supplementation has therefore been reported to be helpful in various immune and brain-related disorders, as it reduces the behavioral scores of rats suffering from neuropathic.

The researchers [7,8] mentioned H.pylori positivity rates seem to be higher among populations with low serum Vitamin D3 Levels, Thus, starting from an association between Vitamin D3 deficiency and various types of infections,an  inverse linear correlationwas established between Vitamin D3 sufficiency and H.pylori infection prevalence, which suggested that adequate Vitamin D3 levels might offer protection against H.pylori. Furthermore, one study conducted in Bahrain highlighted that for each unit decrease in serum Vitamin D3 level the risk of H.pylori infection increases by 1.1,thus Vitamin D3 deficiency has been regarded as one of the potential risk factors for H. pylori therapeutic failure.In addition to the metanalysis done [9] found that Vitamin D3 has a protective role in H.pylori infection. Also, [10] found that low level of Vitamin D3 associated with failure of treatment of H.pylori.while [7] reported that increased Vitamin D3 levels are associated with successful H. pylori eradication of H.pylori infection models in both wild-type and VDR knockdown (VDR-KD) mice, which were used to demonstrate that VitD3 inhibits H.pylori infection by enhancing the expression of VitD receptor (VDR) and cathelicidin antimicrobial peptide (CAMP). In addition , VDR-KD mice that exhibited lower VDR expression were more susceptible to H.pylori infection. In cultured mouse primary gastric epithelial cells, we further demonstrated that the VitD3/VDR complex binds to the CAMP promoter region to increase its expression. These data provide a mechanistic explanation of the anti-H.pylori infection activity of VitD3 at the molecular level in mice and suggest a new avenue for the clinical management of H.pylori eradication therapy, for this,  a link between Vitamin D3 and H.pylori infection has been found that constitutes grounds for further research [11]

Diagnosis of H.pylori is one of the most important steps in infection management, Several diagnostic methods are currently available for detecting H.pylori infection, with both high sensitivity and specificity,Diagnostic tests are classified into non-invasive and invasive procedures,the non-invasive diagnostic tests are the urea breath test,stool antigen test, serological tests and tests using molecular methods [12]. The Stool Antigen Test (SAT) has several advantages, including its  non-invasive  nature, rapidity,  high  sensitivity, specificity,  and dependability, It  can  be used  to diagnose infections as well as to assess the success of therapies,Its  low cost, convenience  of implementation  and capacity  to  gather samples have all contributed to its increasing acceptance,In addition,serological assays detect antibodies  towards H.pylori with  the enzyme immunoassay (EIA) representing  the  most often performed,such  test  evaluates the immunoglobulin G(IgG) and has  sensitivity and specificity  values  ranging from  ‘60%  to 100%’[13] The normal levels of Vitamin D3 in the blood can be classified according to Endocrine Society of Clinical Practice (ESCP) as: Deficiency(Less than 20ng/mL),Insufficiency(21-29 ng/mL ),Sufficiency(More than 30 ng/mL) and Toxicity(More than 150 ng/mL) [14]

The most  materials and equipment used during the research are shown in tabe (1).

Table(1): most materail and equipment used in current study.

Samples collection:The samples was collected from different patients at Ibn Sina General Hospital and Al-Salam Teaching Hospital in the city of Mosul/ Nineveh /Iraq.In addition the samples were taken after obtaining official approval from the Nineveh Health Department as well as with the patients’ consent.

The total number of samples was (120) divided as follows:

A. (60) stool samples.

B.(60) blood samples (about 5 – 10 ml from each person) were collected in disposable gel tubes from the same patients those collected stool samples.

Stool  samples were used for detecting the presence of H.pylori Ag,the samples were collected from patients  by giving each patient sterile stool collection container to place the sample in,with the patient being advised to put a sufficient amount of stool in the container, then the examination was performed in the hospital laboratory directly after collecting the sample.

The assay was performed according to the cassette manufacturer's instructions (PPA:99.69% and NPA:9945%) which includes the following steps:

1.Allow the reagent to equilibrate to room temperature for 30 minute(10-30°C) prior to testing

2.A.for solid samples: The cap of the stool collection tube containing the sample diluent was unscrew, then randomly stap the stool collection applicator into the fecal sample in 6 different sites to collect approximately 50 mg of feces.

B.for liquid samples: Approximately 50 µL of liquid fecal was adding into the stool collection tube containing the sample diluent.

3. The cap was tighten onto the stool collection tube,then the stool collection tube was shaking vigorously to mix thoroughly,then the tube was leaving alone for 2 minutes.

4. The outer packing was take off then the strip/cassette was puting onto the desk with the sample adding region of the strip/sample well of the cassette up.

5.The cap of the stool collection tube was break off ,then drop 3 drops(80µL -100µL) of processed sample vertically into the sample adding region of the strip/sample well of the cassette 

6.The test result was observed immediately within (10-20) minutes.

7. Interpretation of results :the appearance of both the test line (T) and control line (C) within the period-specific (15-20 minutes) designates that the collected sample tested positive for H.pylori infection.The appearance  of only the control line (C) designates a negative test result, If the control line (C) does not appear the result is considered invalid and the procedure needs to be repeated.

As for the blood samples, the samples were collected from the same individuals  whom stool samples were collected by drawing a blood sample and placing it in a gel tube then after separating the red blood cells and plasma from the serum by centrifuge, the serum samples

 was stored at(2-8°C) for less than 24 hours to perform the required tests, which included:

 A. Detect H.pylori Ab by Rapid Test cassette(PPA:99.38% and NPA:99.17%) which includes following steps:

1.The reagent was allow to equilibrate to room temperature for 30 minutes(10-30°C) period to testing.

2.The test strip was removed from the sealed pouch and placed it on a clean and level surface with the sample adding area to up.

3.One drop(25 µL)of serum was added onto the sample adding area of the strip with avoid formation of bubbles,then two drops(80µL -100µL) of the sample diluent was added onto the sample adding area of the strip.

4. The test result was observed immediately within 15-20 minutes.

5. Interpretation of results: The same instructions as in step number 7 when testing H.pylori Ag was followed.

B. Measurement level of Vitamin D3 Which was performed by a VIDAS device which is an automated quantitative device use for the determination of 25-hydroxy Vitamin D Total in human serum using ELFA technique,this test was donig by inserting the SPR_S and strips into the instrument ,then 100 µL of serum was added  to first well of strep and selecting start,all the assay setps are performed automatically by the instrument,then the assay will be completed whithin approximately 40 minutes and finally the results were recorded.

The stander T-tset was used to compared the results between the osteoporosis and control groups.

The current study included the detection of the presence of H.pylori Ag  and H.pylori Ab in stool sample and blood sample  respectively of the same patients, Where it is shown that 53.33% of stool sample were positive for H.pylori Ag test as in figer (1.A) while 46.66% was negative for this test as in figer (1.B) 

Figure 1: H.pylori rapid test. (A) Positive test. (B) Negative test

As for blood samples to detect  of H.pylori Ab the results showed that 55%  and 45% was positive  and negative for this test respectively.the difference btween presence of H.pylori Ab  more than rate of presence of H.pylori Ag in the same individual may result from taking the treatment or the patient's immune system being  able to eliminate the bacteria and H.pylori Ab may remain in the patient's body for a long period and significant reduction in H.pylori Ab titer occurs within one year after eradication treatment, but that a long period is needed to achieve complete negative conversion,this study was consistent with the previous study such as [15] After conducting a blood sample test to measure the level of Vitamin D3, the results showed that 84.37% of the patients  those who positive for H.pylori Ag have Vitamin D3 less than (29) ng/ml,while 6.25% of  H.pylori Ag positive patients have Vitamin D3 more than (30) ng/ml as in table (2).Whereas the patients those  negative for H.pylori Ag and have Vitamine D3 less than (29) ng/ml their percentage was 35.71% and 64.28% have Vitamine D3 more than (30)ng/ml

Table 2: levels of Vitamin D3 in positive and negative samples for H.pylori Ag

The results of the current study were consistent with previous studies when comparing Vitamin D3 deficiency and the presence of H.pylori Ag, among these studies [16,18].Evidence from the recent literature indicates that Vitamin D3 possesses immunoregulatory functions that exhibit an effect on susceptibility to infections in general and to H.pylori specifically, Vitamin D3 might decrease the risk of infection by various mechanisms; Vitamin D3 improves innate immunity by modulating the production of antimicrobial peptides and cytokine response [9].In addition, Several clinical studies have illustrated that Vitamin D analogues may have anti-H.pylori  effects from this study  [19,20]

Our study provides evidence for the relationship between  H.pylori infection with  the level of Vitamin D3, The level of Vitamin D3  in patients  with H.pylori infected is lower than the level of this Vitamin patients not infected to this bacteria,Therefore, a periodic examination to detect the presence of H.pylori  infection is recomended to prevent it^,s complication.

The authors declare that they have no conflict of interest

No funding sources

The study was approved by the university of Mosul, Nineveh Government, Iraq.

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