1. Introduction
Helicobacter pylori (H. pylori), a coiled mobile and microaerophilic, is a Gram-negative bacterium that colonizes the human host stomach, where it causes inflammation and affects gastric physiology. The overall prevalence of H. pylori infection is close to 50 percent, with Africa bearing about 70% of this prevalence, followed by South America at 69.4% and Western Asia at 66.6% [1] . The high rate of Helicobacter pylori, especially in emerging countries, is probably due to the transmission mode, such as direct contact between family members and consuming contaminated food and water [2] . In Burkina Faso, H. pylori rate is high and varies between 80% - 92% according to the study population and the diagnostic method [3] [4] [5] . It is known that gastritis, peptic ulcer disease, mucosa-associated lymphoid tissue lymphoma, and gastric cancer are caused by H. pylori [6] [7] . Gastric cancer represents the twelfth most common cancer in Africa [8] . The bacteria virulence genes have been associated with the latter diseases. Among these genes, cagA is the most frequent and essential of the cag pathogenicity Island (PAI) of the genes linked to H. pylori cytotoxin, it is also an oncoprotein because of its multiple associations with gastric cancer [9] . The Cag (PAI) synthesizes type IV secretion (T4SS), which injects oncoprotein cagA into the host epithelial cells. H. pylori strains expressing cagA have been associated with gastroduodenal ulcers and gastric cancer [10] [11] .
The vacuolating cytotoxin A (vacA) can induce vacuole formation in eukaryotic cells. It is also found in most H. pylori strains. VacA is composed of three (3) main regions: the signal (s1 and s2), intermediate (i1 and i2), and middle region (m1 and m2). It is associated with effects like proliferation inhibition and induction of apoptosis in gastric cells. H. pylori vacuolating activity is linked to vacA genotypes (s1/m1, s1/m2, and s2/m2) [12] . The presence of cagA is often associated with the genotype s1/i1/m1 of vacA [13] .
Additionally, the blood group antigen binding adhesin (babA) is encoded by the babA2 gene. It is located on the outer membrane of H. pylori as a principal adhesin. It identifies as the blood group antigens Lewis b on the host gastric epithelium and characterizes H. pylori colonization density. Its presence correlates with cagA and vacA by increasing infection complications [14] [15] . Furthermore, upon attachment to the gastric epithelia favored by babA, H. pylori expresses virulent proteins cagA and vacA to escape the host immune systems. VacA and cagA can work together, vacA causing autophagy which allows cagA to accumulate in the cells [16] . The importance of Helicobacter pylori in the occurrence of gastroduodenal diseases and gastric cancer, there is a need to provide information on its virulence genes in Burkina Faso in the context of a low-income country. The hypothesis of hygiene and virulence subtypes of Helicobacter Pylori correlation is that virulence genes as part of the bacteria would be transmitted mainly through contaminated food and water. Helicobacter pylori virulence genes enable the bacteria to successfully colonize the gastric mucosa and allow persistent infection, which would cause inflammation and tissue damage. This study aimed to characterize Helicobacter pylori virulence genes and the associated behavioral factors among dyspeptic patients in Burkina Faso.
2. Material and Methods
2.1. Study Population and Sampling
The study population comprised two hundred and fifty (250) patients suffering from dyspepsia. The laboratories of the Saint Camille Hospital and the Pietro Annigoni Biomolecular Research Center (CERBA) were the settings where the patients were consecutively recruited between January and April 2020. A medical doctor prescribed a stool exam suspecting an H. pylori infection. The stool sampled was conserved at −80˚C after being resuspended in DNase-free water.
2.2. DNA Extraction
Bacterial DNA was extracted from stool samples using a commercial kit (QIAamp DNA Stool Mini Kit, Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The DNA quantity and purity were measured using a Biodrop before running the PCR assay.
2.3. Molecular Detection of H. pylori and Its Virulence Genes
CagA, babA2, vacA (s1, s2, i1, i2, m1, m2) virulence genes were detected by multiplex Polymerase Chain Reaction (PCR) with sets of primers per PCR, H. pylori rRNA16s gene using specific primers (Table 1). The latter gene, rRNA16s, was used to confirm the effective presence of H. pylori DNA in the samples. The PCR reaction master mixes were prepared in a final volume of 25 µL containing 12 µL of 1.5x FIREPol® Master Mix, 2 µL of primers (0.5 µL of each sense and antisense primer), 6 µL of sterile H2O and 5 µL of each DNA sample. A PCR program was used for the amplification and consisted of: 94˚C for 5 min, followed by 35 cycles at 94˚C for the 30 s, 57˚C for 45 s, 72˚C for 1 min, and finally, a final extension at 72˚C for 7 min on the GeneAmp PCR System 9700 (Applied Biosystems).
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Table 1. Helicobacter pylori virulence genes sequences.
F: forward; R: reverse; bp: base pair.
2.4. Statistical Analysis
The collected data were analyzed with SPSS version 25 software (SPSS, Inc., Chicago, IL). Two by-to-table statistics and a chi-square test were run to determine associations between H. pylori virulence genes, and behavioral factors. A binomial logistic regression test was also run to appreciate the link between H. pylori virulence genes and some risk factors. A p-value < 0.05 was considered statistically significant.
2.5. Ethics
The study obtained the approval of the Ethics Committee for Health Research of Burkina Faso (Deliberation n˚ 2020-12-274). All participants or guardians of participants gave their free and informed consent. Confidentiality and anonymity of the information provided were respected.
3. Results
The overall prevalence of H. pylori among the 250 participants was 91.2% (228/250). The patients recruited ranged in age from 4 to 80 years, with an average age of 38.56 ± 15 years. The age range of [20] - [40] represented 55.60% of the study population. Women were also the most described in our study (57.60%). Most patients resided in urban areas (96.80%), and the majority were from the informal sector (61.20%). H. pylori were present among 67.36% (97/144) of the women, while it was present among 78.30% (83/106) of the males with p = 0.057.
3.1. CagA, babA2, and vacA Virulence Genes Detection in Stool Positive for Helicobacter pylori
CagA virulence gene was present among 20.19% of individuals, while babA2 and vacA were detected respectively among 9.65% and 67.54% (Table 2).
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Table 2. Prevalence of Helicobacter pylori genotypes typed.
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Figure 1 illustrates the different frequencies of vacA subtypes. Among vacA subtypes, vacAs1 was the most frequent, with 39.04%, followed by vacAi1 (19.74%), vacAi2 (17.54%), and vacAs2 with 10.96%. Regarding vacAm1 and vacAm2, they were less frequent at 6.14% each.
Genotype-wise, vacAm2s1 was the most frequent, with 4.82% (11/228), followed by vacAm1s1 at 3.5% (8/228). Furthermore, vacA genotype m2s1i1 was 2.2% (5/228) while vacAm2s2 and vacAm2s2i2 genotypes frequencies were 1.3% (3/228) each, followed by vacAm1s1i1 with 0.88%. Finally, vacA genotypes m2s2i2 and m1s2 were least frequent, with 0.44% (1/228) (Table 2).
Figure 1. Helicobacter pylori vacA subtypes frequencies in our study population.
3.2. Relation among cagA, babA2 and vacA Subtypes s1, s2, m1, m2, i1 and i2
Table 3 shows the relation among cagA, babA2, and vacA subtypes. We noted that vacAm2 and vacAs1 were highly linked with a p-value = 0.004, while babA2 was linked to vacAs2 (p = 0.027). Additionally, babA2 and vacAm2 were significantly associated, with p = 0.045 (Table 3).
3.3. Relation between Socio-Demographical, Behavioral Conditions and H. pylori Virulence Genes cagA, babA2, and vacA Subtypes Carriage
Helicobacter pylori virulence genotypes association with some socio-demographical and behavioral factors was determined in Table 4. Of the collected information, only the number of handwashing per day was associated with H. pylori infection, highlighted by the presence of the rRNA16s gene, and was statistically significant (p-value = 0.018). Age was associated with vacA subtypes s2 and m2, and the p-values were 0.003 and 0.015, respectively, while sex and area of residency were not associated with the virulence genes in this study. The type of profession was associated with the vacAi1 (p-value ≤ 0.001). The number of handwashing per day less than or over three (3) was associated with vacAi2 (p-value = 0.004). The consumption of fresh products, such as fresh milk, fruits, and raw vegetables, was the most associated factor with the vacA subtypes: vacAm1, vacAm2, vacAi1, and vacAi2 with p-values of 0.040, 0.008, 0.0001, and 0.012, respectively. Alcohol consumption was linked to the babA2, and the p-value was 0.013. The type of water source (running water or not) was associated with the virulence genotypes cagA (p ≤ 0.0001), vacAi1 (p ≤ 0.003), and vacAi2 (p ≤ 0.007). The number of persons per household was associated with vacAs1 and vacAi2; p-values were 0.033 and 0.0001, respectively. Taking the meals alone or in a group was not associated with a virulence genotype.
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Table 3. Relation among Helicobacter pylori virulence genes alleles studied.
3.4. Socio-Demographic and Behavioral Factors Associated with H. pylori Virulence Genes Carriage
Table 5 presents socio-demographic and behavioral factors associated with H. pylori virulence genes carriage. Handwashing equal to or less than three times per day increased significantly by almost four (4) and three (3) times the risk of having vacAi2 genotype and H. pylori rRNA16s, with p-values = 0.013 and 0.020, respectively. Persons who did not consume fresh fruits or raw vegetables had eight (8) times the risk of having the vacAi2 genotype, and it was statistically significant, p-value = 0.008. Furthermore, not drinking alcohol seems to reduce the risk of having vacAi1 and babA2 genotypes by 72% and 66%, and the p-values were 0.018 and 0.051, respectively. Persons who do not drink tap water (running water) have almost seven (7) times the risk of carrying the cagA virulence gene. Additionally, patients with more than four (4) people in their household had about two times the risk of vacA genotype s1, while it was a reduced risk by 67% for those carrying vacAi2 genotype and p-value = 0.006.
4. Discussion
Many H. pylori virulence factors induce infection complications, leading to gastric cancer. In our context, biopsies and an H. pylori culture can be costly and
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Table 4. Link between socio-demographical, behavioral conditions and H. pylori virulence genes, cagA, babA2, and vacA subtypes carriage.
challenging. Therefore, in this study, we searched for Helicobacter pylori rRNA16s and selected virulence factors in stool samples. The overall Helicobacter pylori frequency found in our study was 91.3% which is close to that of Werme et al., in 2015 or Serme et al., in 2016, who found respectively 91.43%, and 87.21% in Burkina Faso [3] [5] . These frequencies are similar to the 97% reported in the Gambia [21] , 93.1% in Congo [22] , 75% in Rwanda [23] , while it was 69.9% in Morroco [24] , and 50% in South Africa [25] , although the studies were not done on the same type of samples. In the literature, Helicobacter pylori are transmitted early in life, especially in sub-Saharan Africa, explaining this high prevalence [4] . The early infection of H. pylori may be why the rates of gastric cancer are low in Africa. This high transmission of Helicobacter pylori is found by many studies to be probably from person to person, as in fecal-oral, gastric-oral, oral-oral, or through contaminated food and water [26] [27] .
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Table 5. Socio-demographic and behavioral factors associated with H. pylori virulence genes carriage.
OR: Odds Ratio.
This study reports a strong association between sanitary habits and Helicobacter pylori’s virulence factors typed. Handwashing increased by three (3) times the risk of having Helicobacter pylori infection, while it was the “number of people in the household the subject grew up with” that was a risk factor in the studies of Smith et al., in Nigeria [28] , and Belay et al., in Ethiopia [29] . The fact that persons did not drink tap water increased by six times their risk of carrying the virulence gene vacA when infected by Helicobacter pylori. However, in Santibanez et al., study, cagA was related to active tobacco smoking in Spain [30] . Not drinking alcohol seems protective of carrying the babA2 virulence gene within our research. Furthermore, we report here that vacAs1, vacAi1, and vacAi2 are significantly associated with the number of people in a household, consumption of fresh products, and the number of times a person washes their hand per day, respectively.
Helicobacter pylori’s pathogenicity island cagA is an oncoprotein due to numerous associations with gastric cancer [9] . We report cagA with a frequency of 20.19% for our study population. This prevalence is surprisingly low compared to that of 74.8% reported in Ghana [31] , 73.3% in Senegal [32] , 52% reported in Gambia [21] , 42.3% in Morroco [24] , and 96.4% in Nigeria [33] . BabA2, another virulence gene associated with gastric epithelial cell adherence, has very few studies in sub-Saharan Africa. Here we report a prevalence of 9.65%, which is very low compared to the 83.3% reported in Cuba [34] and 94.6% reported in Iran [35] . Furthermore, vacA or vacuolating cytotoxin is involved in the progression of gastroduodenal diseases. The gene has a toxigenic effect as it binds to the eukaryotic lipid sphingomyelin receptor; it then targets mitochondria, induces apoptosis, and makes large extracellular vacuoles.
VacA genes have polymorphisms and are structured mainly into signal, intermediate, middle, and deleted regions; its frequency is 67.54%. The vacAs1 allele was the most represented in our study population, similar to the studies from Senegal, Ghana, The Gambia, and South Africa [21] [25] [31] [32] . The allelic combination vacAs1/m1 is the most virulent, whereas s1/m2, s2m1, and s2m2 genotypes show low to no pathogenicity [12] . We report in our study the presence of s1m2 (4.82%), s1m1 (3.51%), s1m2i1 (2.19%), s2m2 (1.31%), s1m1i1 (0.88%), s2m1 (0.44%), and s2m2i2 (0.44%), similarly to Rhead et al., [36] . Overall, the frequencies of cagA, vacA, vacA subtypes, and babA2 were low than those reported by Archampong et al., in Ghana, Breurec et al., in Senegal, and Idowu et al., in South Africa [25] [31] [32] . These differences could be due to the type of the study population, the type of sample used, and the strains of H. Pylori present.
The women were the most represented group in our study population; however, the Helicobacter pylori infection rate was higher among men (78.3%) than women (67.4%), which was insignificant. The gender difference in H. pylori infection was also reported in previous studies by Compaore et al., Replogle et al., and de Martel et al., and the immune system response can partially explain this difference, as women might have protective immunity against H. pylori [37] [38] [39] . Studies imply that immune response differs between men and women [40] . Biologically, estrogen stimulates immune responses, while testosterone is immunosuppressive [41] . Many virulence genes of H. pylori have been associated with peptic ulcer, duodenal and gastric cancer. However, due to incomplete patient data, this study did not use samples from known gastric cancer patients or gastroduodenal diseases. Our research shows the presence of several H. pylori virulence factors in stools, but the link between these factors and Helicobacter pylori-related diseases is yet to be thoroughly investigated. This study is a stepstone that allows clinicians and researchers to know which subtype of vacA is present in our context. This information can be used in research to improve the eradication treatment in a context of antibiotic resistance. It may also help clinicians predict patients at risk for gastric cancer due to their VacA and CagA virulence gene profiles.
5. Conclusion
The present study shows the presence of Helicobacter pylori virulence genes cagA, vacA, vacA subtypes, and babA2 in stool samples by polymerase chain reaction method in Burkin Faso. Additionally, the strong association between sanitary habits and virulence factors typed depicts the composite interaction between ecological factors, gastric mucosa, and bacteria. Therefore, synergic action of these factors needs to be considered when aiming for the bacteria’s eradication and gastric pathology cure.
Authors’ Contributions
TRC and KT conceived and designed the experiments and wrote the manuscript; TRC, KT, NIC, LT, SZ, STS, DK, DS, YAS, and TS performed the experiments; WFG and HGO supervised the research and finalized the manuscript. JS contributed to the study design, experimental assays, writing, and critical reviewing of the content and approved the final version of the manuscript. All authors have read and agreed to the published version of the manuscript.
Data Availability Statement
The data supporting this study’s findings are available upon request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.
Funding
This study was supported by which was supported by “The World of Science Academy” (TWAS, grant: 20-080 RG/BIO/AF/AC), the “Institut de Recherche en Sciences de la Santé”/“Centre National de Recherche Scientifique et Technologique” (IRSS/SCNRST) and the “Laboratoire de Biologie et de Génétique Moléculaire” (LABIOGENE), Université Joseph KI-ZERBO.
Acknowledgements
We thank the technical staff of the “Centre de Recherche Biomoleculaire Pietro Annigoni” for the excellent technical assistance with the sample collection.