Association of Helicobacter Pylori Infection with Cardiovascular Diseases Subjects from Cameroon, Using GastroPanel® Serological Biomarker Panel (Pepsinogen I; Pepsinogen II; Gastrin-17; Helicobacter Pylori IgG)

1 Faculty of Science, University of Buea, Cameroon. 2 School of Assistant Laboratory Technologies, Limbe, Cameroon. 3 Faculty of Health Sciences, University of Buea, Cameroon. 4 Antimicrobial and Biocontrol Agent Unit, University of Yaounde, Cameroon. 5 Sante (Meilleur Acces Aux Soins De Santé), Younde, Cameroon. 6 School of LaboratoryTechniciansYaounde, Cameroon. 7 Faculty of Medicine and Pharmaceuticals Sciences, University of Douala, Cameroon. 8 Department of Clinical Research, Biohit Oyj, Helsinki, Finland. 9 Molecular Oncology Research Center, Barretos Cancer Hospital, Barretos, SP, Brazil.


INTRODUCTION
Endemic non infectious diseases including cardiovascular diseases are among the most deadly diseases of the 21st century [1]. Various risk factors including hypertension, increased lipid level, obesity, diet with highfat, physical inactivity, diabetes, and stress conditions are generally associated with cardiovascular diseases (CVD) [2]. Some microorganisms have been reported to contribute in the development of cardiovascular diseases and are considered as risk factors, amongst them the spiral rodshaped gram-negative bacterium, Helicobacter pylori (H. pylori) [3,4]. Helicobacterpylori is associated withmany gastrointestinal diseases including gastritis, peptic ulcer disease, gastroesophageal reflux disease, chronic atrophic gastritis (CAG) and gastric cancer [5,6]. Helicobacter pylori infection remains high on a global scale with an estimatedof 4.4 billion people infected but differs significantly among populations in various geographical regions [7]. Chronic infection with H. pylori strongly increases the risk of CAG which causes vitamin B12 deficiency that was found responsible for CVD. Because deficiency of vitamin B12 is one of the causes of hyper-homocysteinemia [3,8]. Data on the association of H. pylori and cardiovascular diseases are scarcein Cameroon. We therefore sought to evaluate the prevalence of H. pylori infection in patientswith cardiovascular diseases and also find out it there exist any significant correlation between H .pylori IgG antibodies and biological markers in patients attending the Yaounde Central Hospital.

Patient Information
Patients consulting for various cardiovascular complications and consented to participate were prospectively used for this study between February and May 2019 at the cardiology unit of the Yaounde Central Hospital.

Blood Samples
Basal blood was aseptically collected after at least 4hours of fasting by venipuncture into EDTA tubes and immediately centrifuged at 2000G for 15 minutes. The plasma samples were then distributed into cryo tubes and stored at -20 o C until analyzed. Plasma concentrations of PGI, PGII, G-17 and H. pylori IgG determined by the Gastro Panel (Biohit plc Helsinki, Finland) (9) using the enzyme linked immunosorbent assay (ELISA), according to the manufacturer's instructions for the measurement of absorbance after a peroxidation reaction at 450nm. The results of the GastroPanel® examination were evaluated using the Gastro Soft® interpretation software [9].

Assay Analysis
Based on the clinically-validated cut-off valuesfor each biomarker, the software classifies the test results into one of the five categories: 1) Normal result, 2) superficial gastritis (non atrophic gastritis), H. pylori infection without atrophy 3) atrophic gastritis in the corpus, 4) atrophic gastritis in the antrum, and 5) atrophic pangastritis. The recommended cut-off values were used for all 4 biomarkers as follows: pepsinogen I(PGI) <30 μg/l, the PGI/PGII ratio <2.5, and fasting G-17 <1 pmol/l (G-17b). Values below these cut-off levels implicate AG of the corpus (PGI, PGI/PGII) and AG of the antrum, respectively. H. pylori IgG antibody levels above 30 EIU (enzyme-immunoassay units) were considered as indicator of H. pylori infection.

Statistical Analysis
Statistical analysis was performed using the EPI Info 7.0 software package. Data were expressed as mean±SD. Thedifferences between groups were analyzed by the Student's t-test, Mann-Whitney U-test and Significance of differences between means was estimated with ANOVA, and between proportions using χ2 test. In all tests, values with p<0.05 were regarded statistically significant. Ethical clearance was obtained from the national ethics committee. The study was accepted by the ethics committee of the Yaounde Central Hospital. All patients signed an informed consent form.

RESULTS
A total of 62 patients were enrolled duringthestudy period, aged 30-75 years ( [11] (78.8%). This prevalence is also similar to those previously reported in dyspeptic patients [12] (81.40%), [13] (79.80%) and in diabetic subjects in Cameroon [14] (88.2%). However, our results contrast sharply with those reported by [15] (29.5%) using the PCR test, [15] (23.8%) using antibodies against immunoglobulin A (IgA) and [15] (53.3%) using antibodies against immunoglobulin G (IgG). This may be as a result of diagnostic technique used. Several possible mechanisms and pathways have been described as to how H. pylori contribute to cardiovascular complications [10]. Repeated exposure to infection leads to failure of the inflammatory process and inability to control the progress of infectious agents that leads to a number of diseases such as heart disease and cancer [16,17]. Chronic stimulation due to bacterial infection in the gastric mucosa produces more induction of dyslipidemia, increases fibrinogen levels, stimulates the release of reactive protein and hyperhomocysteinemia leading to artery blockage and the development of heart problems [3]. Also, chronic infection with H. pylori is known to increase pH levels, increase gastric juice and decrease ascorbic acid levels, which will cause a reduction in folate absorption and thus increase the concentration of homocysteine that causes endothelial cell damage [18]. In addition, atrophic gastritis and hypochlorhydria observed in these subjects are a risk of enteric infections, low absorption of ATBs, vitamin B12 and some divalent micronutrients, including iron, calcium, magnesium and zinc, with an increased risk of clinically important sequelae, such as cognitive disorders, neurodegenerative and vascular disorders, encephalopathies, anemia and osteoporosis [19,20].

CONCLUSION
The study indicates that H. pylori infection is highly associated with various cardiovascular complications and disease risk factors. Thus, patients suffering from various cardiovascular diseases are at risk of gastric cancer and require continuous monitoring.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT AND ETHICAL APPROVAL
Ethical clairance was obtained from Center Regional Committee for Research on Human Health (CRERSH). An authorization was obtained the authorities of the Jamot Hospital of Yaounde. All patients signed an informed consent form.

AVAILABILITY OF DATA AND MATERIALS
All data used during the current study are available from the corresponding author on reasonable request.