Potential risk factors and prevalence of Helicobacter pylori infection among adult patients with dyspepsia symptoms in Cameroon

Background Helicobacter pylori is a Gram negative bacterium that colonizes the stomach of approximately two-thirds of the human population and it is involved in the pathogenesis of gastroduodenal diseases. This study sought to determine potential risk factors associated with seroprevalence of H. pylori among dyspepsia patients in Cameroon for a better management of the disease. Methods The study was carried out from August to December 2014 at Laquintinie Hospital and District Hospital of Bonassama in Douala metropolis. 205 patients (127 women and 78 men; mean age, 53.79 ± 11.11 years; range, 35–75 years) were enrolled. Each subject gave a written consent. The study was approved by the local Ethical Committee of Medical Sciences. A structured questionnaire was used to collect information on sociodemographic parameters and predisposing risk factors for Helicobacter pylori infection. For each patient, body mass index (BMI) and direct inquiry about dyspeptic symptoms were done. Blood samples were tested for H. pylori antibodies, and ABO/Rhesus blood group antigen typing was performed. Results The overall prevalence was 64.39%. All patients with upper abdominal pains and frequent burping were H. pylori seropositive. We found that infection takes place early in childhood and adolescence, and reaches its peak at adulthood at 35 to 44 years. Sixty-two percent of women over 68 of men were infected. 80.39% of patients with family history of gastric cancer were seropositive, while 19.60% were seronegative (p = 0.001). Prevalence of 79.09 and 47.4% was recorded respectively for subjects with low, middle and high income levels (p = 0.001). H. pylori infection rate was 60.48% in blood group O patients compared with 70.37% in other blood groups (p = 0.203). 73% of infected subjects over 59% of uninfected ones currently take NSAIDs (p = 0.0509). Overcrowded households have a higher rate of 65.32% seropositivity in contrast with a lower rate of 33.33% from norm household (p = 0.197). 69.29% of obese and overweight patients versus 58.24% of subjects with normal weight were seropositive (P = 0.215). Conclusion The results of this study demonstrate that low income, family history of gastric cancer, clinical symptoms of nausea/vomiting and flatulence/bloating were risk factors of H. pylori infection in this population.


Background
Helicobacter pylori (H. pylori) is a Gram-negative rod bacterium which lives in the human gastric milieu. Helicobacter pylori colonize the stomach and provoke a local inflammation in almost all host, a continuous process increases the risk of developing atrophic gastritis, intestinal metaplasia, and noncardia gastric adenocarcinoma [1][2][3]. The fourth most common cancer is gastric cancer caused by Helicobacter pylori and it is the second cause of mortality due to cancer in the world [4]. About 50% of the world population are infected by Helicobacter pylori and this rate of infection is higher in underdeveloped countries than in developed countries [5]. The report of the infection rate are as follow, 15.5% for developed countries and 93.6% for underdeveloped countries [6][7][8]. H. pylori is contagious, although the exact route of transmission is not known [9]. A number of authors have emphasized the role of factors such as age, socio-economic status, poor hygiene/deficient sanitation, density/ crowded living conditions, smoking, use of a nonsteroidal anti-inflammatory drug (NSAID), blood group O, high body mass index and family history of gastric disease in the acquisition and transmission of H. pylori [10][11][12][13][14].
Limited information is available on the prevalence of this pathogen among apparently healthy and symptomatic children in two regions of Cameroon: prevalence of H. pylori among apparently healthy children in the Buea and Limbe health districts of Cameroon [15], prevalence of H pylori among children and adolescents from the age range of 6 to 18 years old with peptic ulcer disease in Yaounde [16]. But these studies were practiced on children of age less than 18. The absence of these data in our adult population has hinder the better understanding of the harmful effect of the disease in our society. Also, it slow down the planning for resources which can enable us to best fight against Helicobacter pylori associated diseases which are frequent in our adulthood. Moreover, the roles of potential risk factors associated with the infection have not been elucidated in those studies. As a result, guidelines and other adequate information on the prevention and control strategies of Helicobacter pylori infection are lacking.
Taking into account this information, studies have been initiated by us to elucidate this question in order to bridge the gap in knowledge about the prevalence of infection among adults with dyspepsia symptoms in the littoral region of Cameroon and a thorough knowledge of risk factors such as socio-economic status, poor hygiene/deficient sanitation, density/crowded living conditions, smoking, use of a nonsteroidal anti-inflammatory drug (NSAID), blood group O, body mass index and family history of gastric disease that predispose to infection among this population in Cameroon. This region was chosen because it is the most popular and economic region of the country with high promiscuity, poor sanitation and household hygiene; factors that favor the spread of H. pylori.

Study area
The study was conducted in Douala metropolis, the largest city of Cameroon and the capital of the Littoral Region. Douala is located on the banks of the Wouri River in an area covering about 210 km 2 divided into seven districts and has more than 120 neighbourhoods [17]. Douala typically features warm and humid conditions with an average annual temperature of 27.0°C , an average humidity of 83% and an average annual rainfull of 3600 mm [18,19]. The city and its surrounding area has an estimated population that surpasses 3,000,000 inhabitants. It is the commercial and economic capital of Cameroon and the entire CEMAC region. It has modest oil resource in the world and very good agricultural viability which attract people from many other parts of the country. People from other countries in the region and expatriates have also permanently settled in the city due to its highly developed infrastructures and peaceful environment for successful business. Even though Douala is the economic center of Cameroon, a large percentage of its inhabitants live below the poverty line due to its steadily increasing population. Recent data shows that about 30 % of the population lives in poverty while the aforementioned percentage is doubled for rural zones [17].

Selection of subjects
The study was carried out within the Littoral Region using two public hospitals in Douala metropolis, Laquintinie Hospital and District Hospital of Bonassama. From August to December 2014, all patients aged 35 years or older with the symptoms of dyspepsia or other symptoms referable to the proximal alimentary tract, attended Gastroenterology Department at the selected health center were referred for serologic diagnosis of H. pylori infection. We employed a consecutive sampling for data collection, requesting consent from all volunteer patients in the selected health facilities who fulfilled the eligibility criteria for the study during the study period. Pregnant and breastfeeding women were excluded from the study. The number of patients enrolled was not based on a pre-selected statistical power.

Variables
The following information were requested from the subjects in a structured questionnaire; age, sex, native region, socioeconomic class or income level [low income (≤ 2500 $/month) taking as a reference, Middle income (2500-8500 $/month) and High income (≥ 8500 $/month)], living conditions during childhood (childhood in rural or in urban zone), household population or Number of members of family that sleep in a room (household population ≤ 4 taking as a reference, household population between 5 and 7, and household population ≥ 8), smoking, Non-steroidal anti-inflammatory drugs (NSAIDs) consumption and relatives of gastric cancer. For all participants, direct inquiry about dyspeptic symptoms: upper abdominal pains (epigastric pains, burning), abdominal discomfort (flatulence, bloating), nausea/vomiting and frequent burping was done by a resident gastro-enterologist and the clinical characteristics were recorded. Height and weight were measured for each participant, and BMI was calculated as weight/ height 2 (kg/m 2 ). Participants were classified relative to BMI value in 3 groups; normal (< 25 kg/m 2 ), overweight (25 to 30 kg/m 2 ) and obese (> 30 kg/m 2 ). The participants with normal BMI were taken as reference group for statistical analysis.

Collection of sample/analysis
Three milliliters (3 ml) of venous blood was drawn from each patient, then 2 ml of it transferred into a clean test tube and allowed to clot naturally by standing the sample at room temperature for 10 mn. Clear serum sample was obtained by spinning the tubes at 3000 rpm for 5 mn and used for the detection of antibodies against H. pylori using One Step H. pylori test device (DiaSpot H. pylori, Indonesia). The remaining 1 ml of the blood was emptied into disposable tubes, and used for determination of ABO blood group antigens by a standard hemagglutination test. Participants were classified relative to ABO blood group antigens in 4 groups (Group A, Group B, Group AB, and Group O was taken as reference group for statistical analysis) and in positive or negative according to the rhesus status.
For each sera collected, 3 drops were used to detect Helicobacter pylori antibodies according to the manufacturer's instruction. DiaSpot R H. pylori One Step Test Device is a rapid chromatographic immunoassay for the qualitative detection of H. pylori antibodies in serum or plasma. It is a test that utilizes a combination of H. pylori antigen coated particles and anti-human IgG to qualitatively and selectively detect H. pylori antibodies in serum or plasma. Anti-human IgG is immobilized in the test line region. The sample is added to a specific area on the test device, it reacts with H. pylori antigen coated particles in the test. This mixture migrates chromatographically along the length of the test and interacts with the immobilized anti-human IgG. Appearance of a single red line indicates a negative result while double lines indicate positive sample. This test has sensitivity > 95.9% and specificity about 75.9% with overall accuracy of 85.2% as compared with culture/histology of endoscopic specimens for H. pylori.
The ABO blood group was determined for each patient by the conventional hemagglutination test using the anti-A, anti-B and anti-D sera. The ABO blood grouping procedure is based on the principle of agglutination or clumping as the patient's blood is reacted with anti-A, anti-B and anti-Rh antibodies separately.

Statistical analysis
The data generated were coded, entered, validated and analyzed using SPSS 23. We tested for association in categorical variables using the chi-squared test, reporting corresponding p-values. In case of small numbers in a given group (< 5), the Fischer's exact test was used, and the corresponding p-value reported. For the purpose of hypothesis testing, a control group constituted by uninfected participants was used to compare the strength of the association between reported risk factors and infection with H. pylori.
The odds ratio and the corresponding 95% confidence intervals (95% CI) were used to summarize the strength of association between specific binary exposure and outcome variables. The level of statistical significance for the study was set at p < 0.05. A multivariate logistic regression analysis was done for potential risk factors with p value less than 0.05 or near 0.05 to confirm the variable as risk factors of H. pylori infection. The outcome measure was the detection of the presence of H. pylori antibodies in the serum of patients with gastric-related morbidities, and their association with known risk factors for infection.

Sociodemographic and clinical characteristics of the study population
A total of 205 patients were enrolled. Their mean age was 53.79 ± 11.11 years (range 35-75 years), and the age group from 55 to 64 years the most represented (35.6%; 75/205). With 78 male and 127 female subjects, the overall male/female ratio was 1:1.7. All the 10 regions of Cameroon were represented in our sample population; 60% from the West Region, 25.36% from the Littoral, 12.20% from the Center, East and South, 1% from the South and North West, 1% from the Far North, North and Adamawa. 0.5% of the patients were from others countries. Fifty four percent (53.65%; 110/205) of the subjects were unemployed (low income or poorly skilled class) followed by 26.34% (54/205) from middle class with 2500-8500 $/month and 20% (41/205) from elite class with relatively high income (≥ 8500 $/month). Half (102/205) of our sample were living in rural zone during childhood. A similar distribution was recorded as far as family history of gastric cancer was concerned. A household of three to four in a Cameroonian standardized room is considered as the norm; households that are crowded have 5-7 people, and the overcrowded, up to 8 people. In our sample, 70% (143/ 205) of households were overcrowded with 8 to 15 people, followed by crowded ones with 27.31% (56/205). Few number of our population currently smoke and take NSAIDs with a frequency of 17.05% (35/205) and 38.04% (78/205) respectively. The distribution of the ABO blood groups among patients was 60.5% for O group, followed by B (18.54%), A (17.56%) and AB (3.41%), while 203 (99.02%) of patients were rhesus positive and 2 (0.975%) rhesus negative. Among the 205 participants, 4.88% (10) and 50.73% (104) were respectively obesed and overweight, while 44.4% (91) were with normal weight (Table 1).
Direct inquiry about dyspeptic symptoms was done on all the 205 enrolled patients and all of them presented at least two of the clinical characteristics. On the clinical signs, epigastric pain/burning and frequent burping were the most common (100%; 205/205), followed by abdominal discomfort (104/205) and nausea/vomiting (49/205) ( Table 2).

Seroprevalence of H. pylori infection of the study population
205 patients with various gastroduodenal symptoms were tested for H. pylori infection using a rapid chromatographic immunoassay for the qualitative detection of antibodies against H. pylori. Of these patients, 132 were H. pylori positive, giving an overall prevalence of 64.39% ( Table 2).
As regards the clinical signs, all H. pylori (100%; 132/132) infected and uninfected patients presented epigastric pain/ burning and frequent burping as dyspeptic signs, thus these two symptoms cannot be used to predict the etiology of epigastralgia as H. pylori-related in our sample population. However, when considering the other clinical signs evaluated, we noticed that infected patients were prone to develop Flatulence/ bloating (70.45 versus 15.07%) and Nausea/Vomiting (34.1 versus 5.48%) than uninfected ones  The strength of the association between sociodemographic parameters evaluated and infection with H. pylori, shows different rates of infection relative to the age group and sex of the participant, but this difference was not statistically significant ( Table 3).
The highest prevalence was obtained in the age group of 75-84 (80%), but the number of patients aged ≥75 years studied, has been much smaller than those younger than 75 years. So, we found that frequency of seropositivity for H. pylori seems to be at the same level among participants aged over 35 years. Comparing seropositivity in males and females, our study confirms the fact that more males (67.94%) are being seropositive than females (62.20%). But the difference was not significant (P = 0.4044).  The association between H. pylori infection and the potential risk factors are summarized on Table 4. Contrary to smoking and living conditions during childhood which did not affect H. pylori status, the other risk factors evaluated have a positive effect on the frequency of H. pylori infection, with a higher risk of being seropositive among exposed patients than unexposed ones, but the strength of the association was only statistically significant with the income level and family history of gastric cancer ( Table 4).
Similarly, socioeconomic status of participants seems to play an important role on the frequency of infection in this study. Prevalence of 79.09% in unemployed subjects with low income was recorded, in contrast with lower rate of 47.40% from subjects with middle and high income. This difference was significant (p = 0.001).
A multivariate logistic regression analysis was done for the variables; income level, NSAIDs consumption and family history of gastric cancer. The results are summarized on Table 5. From this table, we noticed that among these three categorical variables, income level (p = 0.001) and family history of gastric cancer (p = 0.002) were still associated to H pylori infection with a statistically significant strength.
As ABO blood group status is concerned, our data showed that, among the 124 participants with type O blood group, 75 (60.48%) were seropositive, while 49 (34.50%) were seronegative (Table 4). H. pylori infection  (26/38) in group B, and 71.42% (5/7) in group AB, which were slightly higher than blood group O, but without statistical significance (Table 1). Regarding rhesus (Rh) status, 64.03% Rh (+) and 100% Rh (−) patients were positive for H. pylori. Although Rh (−) patients in our sample seemed to have high seropositivity, this blood group was not representative because it was not common in the study population.
We also noticed that overcrowding increases the chance of seropositivity among the households population, although this difference was not significant (P = 0.197). Households with > 4 members have a higher rate of 65.32% seropositivity in contrast with lower rate of 33.33% from household with ≤4 members (Table 4).
Our data demonstrated that obesity and/or a high BMI is associated with an increased incidence of H. pylori infection. A seropositive rate of 69.29% (79/ 114) was recorded among obesed and overweight patients, in contrast with a lower rate of 58.24% from subjects with normal weight. However, this difference was not significant (P = 0.215).

Discussion
The diagnosis of gastric infection with H. pylori usually involves upper endoscopy. However, serological methods have reached sufficient accuracy to be used as screening tests before endoscopy, or for sero-epidemiological surveys as individuals infected with H. pylori develop antibodies which correlate strongly with histologically confirmed H. pylori infection [20][21][22][23][24].
In our study we evaluated the seroprevalence of Helicobacter pylori infection among patients aged 35 years and older with dyspepsia symptoms in Douala metropolis, and we found a seroprevalence of 64.39% which is lower than the prevalence of 92.2 and 79.3% reported respectively by Ndip et al. (2004) [15] in the North-west region, and Andoulo et al. (2015) in the Center region of Cameroon [16], both among children less than 18 years old. The age range difference in the studied population between the present and these previous studies, may explain such observation. In fact, in our sampling processes, we included only patients aged 35 years or older, who are thought to better observe principles of cleanliness compared to younger ones. Thus, the incidence rate among patients aged ≥35 years would be more or less lower than that in the younger population. Moreover, the differences in these value can also be due to the study area, the socioeconomic conditions in the study area and the type of controls used, since the prevalence of Helicobacter pylori is known to represent the improvements in socioeconomic conditions and sanitary standards throughout the generations. In Russian for instance, within a period of ten years (from 1995 to 2005), it was observed that the prevalence of Helicobacter pylori infection has reduced remarkably due to better standards of living [25]. A similar situation was observed in China due to an increase in economic growth, and improvement in environmental and hygienic conditions [26]. The seroprevalence we found correlates with the results of some studies done in other developing countries within and out of Africa. A seroprevalence of 62. 4% in the Democratic Republic of Congo [27], 50.6% in the North of South Africa (Venda) [28], about 58% in Guatemala [29], 68% in Turkey [30], 61% in Saudi Arabia [31], 62% in Kuwait [32], 66.7 to 85% in some regions of Iran [33,34], and 61.7% in Brazil [35] was observed among the examined individuals.
However, our prevalence was found to be low as compared to those of other studies. For instance, an infection rate of 93% was observed in Ethiopian on patients with peptic ulcer disease [36]. Also, an infection rate of 97 and 94.5% were reported in Ghana [37] and in Mozambique [38] respectively. A prevalence of H. pylori antibodies in an interval of 67 to 84% was found among children in South Africa (Bloemfontein) by Pelsar et al. (1997) [39]. Asymptomatic individuals in Eastern Cape Province were studied and 86.8% of Helicobacter pylori antigenemia was observed in their stools [12]. Ghose et al. (2005) [40] in Venezuela also found a high prevalence of 95. 3% in their study population. The differences between the prevalence of the present study and the ones mentioned above may be as a result of different detection method and geographical area which could reflect the environmental and personal hygiene.
The relationship between clinical signs of the study population and H. pylori status shows that, unlike Upper abdominal pains and frequents Burping, Flatulence/ bloating and Nausea/vomiting are significantly suggestive of H. pylori infection in the study population (Table 5). However, a meta-analysis reported that children with upper abdominal pain or epigastric pain were two to three folds more infected to H. pylori than children without these symptoms [41]. The age range difference in the studied population between the present study and this meta-analysis may explain such observation. In fact, in our sampling processes, we included patients aged 35 years or older and they were considered to be exposed to other etiologic agents of epigastralgia such as NSAIDs abuse than younger population.
Our data on gender agrees with some studies indicate that the males were associated with a higher risk of acquiring H. pylori infection than the females [15], because the males are naturally more active and less hygienic than females, since the prevalence of H. pylori infection and sanitation condition are inversely related. However, some documented studies disagree with this idea, but the reasons is still not known [42,43].
Most infection cause by Helicobacter pylori are highly related to poor living conditions in childhood [44] and in the absence of antibiotics therapy, Helicobacter pylori may persist for life in the host [45]. According to the age group in our study, the newly acquired Helicobacter pylori infection had occurred during childhood and early adolescence, in adulthood (35 to 44 years), it increases and reaches a peak and remains almost the same as you grow older. The present result was in accordance with many other authors' research output. In a rural village of Linqu Country, Shandong Province, China a study on 98 children was made, 70% of those aged between 5 and 6 years were found to be infected with the organism and a similar rate was observed on adult in the same area, so it was concluded that most infection occur at childhood [46].
As regards socioeconomic and family history of gastric cancer status of participants, our data shows that those two factors were significant risk factors for H. pylori infection even after a multivariate logistic regression analysis. We noticed an increase of seropositivity rate from higher class to low or poorly skilled class, suggesting a dreadful trend of H. pylori infectivity due to poor socioeconomic status of participants. This is similar to reports from other parts of the globe, indicating association of low socio-economic factors with prevalence of H. pylori infection in Asia [47], South America [48] and Africa [49]. In fact, poverty enhanced level of transmission due to malnutrition, poor hygiene and unaffordable heath care. Similarly, we also noticed that participants with family history of gastric cancer had a greater tendency towards H. pylori infection. Other studies showed that family history of peptic disease and gastric cancer was significantly associated with H. pylori infection [50,51].
Our finding shows that, the prevalence tended to be higher if there were more family members in a household, although this difference was not significant (Table  5). This confirms the works of several authors that overcrowding facilitates transmission of H. pylori [52,53]. Some other studies have reported household crowding and bed sharing among different people as strong and independent risk factors for H. pylori [54,55]. This is because household members share eating utensils, live in poorly ventilated rooms and are more likely prone to poor hygiene.
Gastroduodenal diseases are mainly caused by Helicobacter pylori infection and the use of a nonsteroidal anti-inflammatory drug (NSAID). A meta-analysis [56] that included 463 studies evaluating the association between H. pylori infection and NSAID used, revealed a higher rate of peptic ulcer disease among NSAID user when Helicobacter pylori infection was present (41.7%) as compared to users who were H pylori-negative (25.9%) [56]. Peptic ulcer disease was 3.5 times more present in NSAID users who were H pylori-positive [57].
In accordance with what was said previously, seropositivity increases with the use of NSAID among our study population. The complementary effect between the two causative factors which lead to the development of gastroduodenal diseases may take place through the production of oxygen reactive species (ROS). In fact, oxygen reactive species favors the development of pathogenesis in acute experimental gastric lesions induced by NSAIDs [57]. Moreover, experimental results strongly support the implication of ROS in the pathogenesis of the H. pylori-induced chronic gastritis [58].
Our finding showed that blood group O was not associated with higher H. pylori infection rate. H. pylori infection rate was 60.48% in blood group O patients compared with 70.37% in other blood groups (p = 0.203), ( Table 4). The H. pylori infection rate in other blood groups were 72.22% (26/36) in group A, 68.42% (26/38) in group B, and 71.42% (5/ 7) in group AB, which were slightly higher than in blood group O, but without statistical significance. The lower rates of other blood groups in the H. pylori infected patients (n = 132) simply reflect the lower prevalence of these blood group types in the study population (Table 1). Previous studies suggested that blood group O do not represent a risk factor for Helicobacter pylori infection [30,59] which agree with our result but disagree with other studies who tie on the fact that individual of blood group O have a higher incidence of developing duodenal and gastric ulcers [43,[60][61][62][63]. This increase in the development of the disease in the blood group O individuals may be due to a great colonization of their epithelial cells and higher inflammatory responses to H. pylori [2].
According to Cohen et al. [64] study, higher BMI levels are common in Helicobacter pylori infected adults despite the fact that the subject may be asymptomatic or not and further suggested that eradication of Helicobacter pylori may lead to loss in weight. Similarly, our study has demonstrated that the probability to Helicobacter pylori infection increase with high BMI level. Some data show that Helicobacter pylori infection can affect the production of leptin and ghrelin and subsequently their plasma level (increasing plasma ghrelin and decreasing leptin levels) [65][66][67][68][69]. The fluctuation in the plasma level of this hormone could promote obesity. In fact, Ghrelin decreases energy loss and promotes weight gain [70], whereas leptin reduces food intake and increases energy loss [71]. In contrast, other studies show that there is no relationship between H. pylori seropositivity or CagA antibody status and high BMI [72,73] and not even an inverse relationship between morbid obesity and H. pylori seropositivity [74].

Conclusion
Our study shows that the prevalence of H. pylori among our sample population is high and that low income, family history of gastric cancer, clinical symptoms of nausea/vomiting and flatulence/bloating were risk factors of H. pylori infection in this population.