Association Between Seropositivity of Helicobacter pylori Infection and Anaemia Amongst Children Aged 5-15 Years in Western Highlands of Kenya

Anaemia is a global health problem, whose development is influenced by several factors. Helicobacter pylori infection is among the factors associated with anaemia. Long term interruption of malaria transmission in the highland area of Kipsamoite led to an increase of haemoglobin levels in some few individuals. However, there are still reported cases of low Hb levels associated with anaemia. In studies that have been done on the role of H. pylori infections on anaemia, researchers hold varied views on whether or not H. pylori infections play a role in iron deficiency anaemia. This study investigated the association between H. pylori infections and anaemia in children aged 5-15 years in Western highland region of Kenya. The study adopted a case-control design and purposive sampling technique was used in selection of subjects. Haemoglobin levels and H. pylori occurrence were measured and entered into Microsoft excel. These data was summarized into descriptive statistics. Chisquare was used in hypotheses testing. A total of 105 participants were enrolled, 35 were cases and 70 were the controls. The mean haemoglobin level was found to be statistically significantly (p<0.00), haemoglobin of cases was lower (11.05 ± 1.726) than that of controls (13.01±1.897). The proportion of anaemic children among the cases was 77.1% (27) while the non-anaemic were 22.9% (8). The Chi-square showed positive statistical significant difference (p<0.05) between cases and controls. The study concluded that there was a great association between H. pylori infection and occurrence of anaemia in Western region of Kenya.


Introduction
Anaemia is still a public health problem globally. According to Benoist, Mclean, Egii, Cogswell (2008), about 293.1 million children under five years are suffering from anaemia with 29 percent being from sub-Sahara Africa. In tropical and developing countries, aneamia is particularly more than 50% prevalent among pre-school children and pregnant women who are either moderately or severely anaemic. Furthermore the main causes of anaemia in tropical countries include: malnutrition, parasitic, bacterial infections, viral infections, inherited haemoglobinopathies, glucose 6 phosphate deficiency and obstetrical complications that result in blood loss (Monica, 2000)In addition, anaemia can be categorized as either, iron deficiency anaemia, megaloblastic anaemia, haemolytic anaemia, anaemia of chronic diseases or a plastic anaemia. According to WHO (2011), iron deficiency anaemia is the most prevalent type of anaemia globally.
Moreover, gastrointestinal bleeding and malabsorption are also factors considered to be contributing to iron deficiency anaemia. Bacteria Helicobacter pylori,is an important microorganism which has been associated with gastrointestinal bleeding and malabsorption and hence considered an important bacterium in iron deficiency anaemia.
H. pylori is a spiral shaped bacterium propelled by flagella, lives in the stomach and small intestines of the host (Monica,2000).Most physicians often overlook these bacteria, even when patients complain of symptoms that are classic for H. pylori infection, which include stomach pain, nausea, acid reflux, intestinal discomfort and halitosis (IDI, 2002).H. pylori needs iron to survive and has been characterized has one of the microorganisms capable of getting heme in hemoglobin and from iron bound to lactoferrin normally found in human secretions such as saliva, tears and gastrointestinal fluids. With an adequate supply of iron, H. pylori can thrive and proliferate to the extent of causing stomach cancer (IDI, 2002). H. pylori utilizes iron for its survival and that is why most studies done, associate this bacterium with iron deficiency.
It has been speculated that H. pylori contribute to iron deficiency anaemia by competing with the host for iron. In addition, Kearney, (2005), points out that colonization of H. pylori in the gastrointestinal tract impairs the absorption of iron and hence affects the ferritin stores causing iron deficiency anaemia without necessarily destroying the gastrointestinal mucosa. The epidemiological studies which have been done have shown that H. pylori is highly prevalent in developing countries than in developed countries.

MATERIALS AND METHODS
The study was carried out in Nandi County, Mosop Sub-County, Sangalo location specifically Kipsamoite sublocation. This highland area has an altitude of 2319M above the sea level, located at latitude of 0.33265N and longitude of 34.99659E. This area experiences low and seasonal malaria transmission within the year, with peaks of malaria outbreaks between the months of May and August. This is influenced by the general climate which comprises of fluctuation in the amount of rainfall between the years and the change in temperature patterns. The area receives high rainfall between the months of March and November with the highest rainfall recorded in the month of August. In different months the changes in minimum and maximum temperature is observed with the highest temperature being noted in the months of January and February. Soil is fertile and supports production of various crops such as maize, potatoes, beans, millet and horticultural crops with tea being the main cash crop. Livestock farming is also practiced for both beef and dairy.
Respiratory tract infections are commonly experienced in this area according to unpublished clinic records. More importantly anaemia, malaria and other bacterial infections are among the recorded and unpublished reports.
The study adopted a retrospective case-control research design involving the study of cases with H. pylori infection compared to controls without H. pylori infection. In addition a cross-sectional study was also adopted, focusing on children between the ages of 5-15 years.
The target population involved children between the age of 5-15 years which presented signs and symptoms of H. pylori infection. Inclusion criteria involved Children who presented with signs and symptoms of H. pylori infections whereas children with an obvious case of blood loss such as active or recent gastrointestinal hemorrhage, those with known chronic hematological diseases other than anaemia, children on antibiotics treatment and those who did not consent to the study were excluded. A total of 105 participants , 35 cases and 70 controls were involved in the study. Sample size was determined using the Kesley formula (Kesley, Whittemore, Evams, Thompson, 1996).
Purposive sampling technique was used to select cases by testing for H. pylori infection in those individuals with signs and symptoms of H. pylori infections, and positive individuals were treated as cases. The controls of the study were obtained, from the participants with signs and symptoms and tested negative for H. pylori infection. Adapting Crimes and Schulz (2005) information, the study recruited 2 controls per case to increase the power of the study.
Collection of Blood Samples involved cleaning the finger with 70% alcohol swab and allowed to dry before being punctured using sterile lancets to enable blood specimen collection.
After collection of blood specimen, Hb level testing was done using photometry (hemocontrol machine) and the readings recorded. Hb levels values was adjusted by -0.8 g/dl for altitude as per World Health Organization recommendation (2011). In addition, the cut off for anaemia was done in relation to the age of the children and this was done as follows: age 5-11.9 years was 11.5g/dl and age 12-15 was 12g/dl. Those children who were found to be anaemic and with H. pylori infections were referred to the health centre clinician for further evaluation and treatment. Serum specimens were collected from the study participants and qualitative detection of IgG antibodies against H. pylori infections was carried out and the results recorded. Children with IgG antibodies were referred back to the clinician for further evaluation.

Data Analysis
Data was entered into Microsoft excel transferred into stata data editor. Descriptive statistics was used, summarized into frequencies (%) and mean (+SD) as appropriate. Chi square test were used in the analysis of the data. Stata (statase14) software was used.

Ethical Considerations
This study commenced after ethical approval by ethical review committee of the University of Eastern Africa Baraton (UEAB/12/9/2017). In addition, permission to conduct the study was obtained from the health centre administration. Further, written informed consent was obtained from the guardian or the parent of each participant and the results were kept confidential. Any result that was necessary for the child was communicated to the physicians for appropriate management. In this study there were 105 study participants (35 were cases while 70 were the controls). Among 35 cases 21 (60%) were female and 14 (40 %) were males. 16(45.71%) were between the ages of 5-11.9 years while 19(54.29%) were between the ages of 12-15 years (Table 1). On the control groups 37 (52.86%) were male and 33 (47.14 %) were female. 30 (42.86%) were between the ages of 5-11.9 years while 40 (57.14 %) were between the ages of 12-15 years ( Table 1). The mean age of cases was 11.586 ±3.7 and that of the controls were 11.41± 3.4. The median age of case and controls were 13 and 12 respectively (Table 1). The median of the Hb of the controls was 13.35g/dl while that of the cases was 11.1g/dl ( Table 2 ). The lowest Hb levels between cases and controls were 6.6g/dl and 8.6g/dl respectively (Table 2). The data values range from 6 g/dl to 16g/dl for both cases and controls (Fig 1). The data of both cases and controls were symmetric. The inter-quartile range of the controls was 3 while the inter-quartile range of the cases was 2 (Fig 1)  In 70 controls, 51 had no anaemia while 19 were aneamic, while in 35 cases 8 were non-anaemic and 27 were anaemic (Table 2). Using pearson chi-square a significance difference was noticed (23.6966, p-value=0.00)

Discussion
Anaemia is one of the public health problems among the children globally. Understanding various factors contributing to development of aneamia helps the clinicians and policy developers to put in place measures on how to deal with anaemia. This study was carried out in area of low malaria transmission with prolonged interruption of malaria transmission in 2008, significant number of cases of anaemia with unknown cause was still being reported even with unreported cases of malaria. The results of this study showed that those children with H. pylori infections had lower Hb levels compared to those children with IgG negative for H. pylori infection. The low Hb levels observed in this study could be due to coupled rapid growth of children and the H. pylori infection. The rapid growth of children requires high amount of iron to sustain their growth (Choe, Kim, Hong, 2000). The development of Hb molecule requires iron and hence any interference of iron affects the Hb levels. Choe et al., (2000) proposed that H. pylori infection affects the iron metabolism and iron absorption and exacerbate the iron deficient. Different researchers have demonstrated various mechanisms which H. pylori could be using to interfere with iron metabolism and iron absorption causing anaemia. The current study postulated that hemorrhagic gastritis associated with H. pylori infection could be a possible mechanisms causing gastrointestinal bleeding causing excess iron loss. Yip, Limburg, Ahlquist (1998) reported that hemorrhagic gastritis associated with H. pylori infection as a possible cause of occult bleeding and therefore iron loss experience in Alaska population.
Secondly, the malabsorption of non-haeminic iron reduces the amount of iron in the circulation affecting the haemopoesis process and hence affecting the level of Hb concentration. Perri et al., (1997) and Windle, Kelleher,Crabtree,(2007) showed that hypochloryhydria in which H. pylori infection is associated with iron deficiency and anaemia, decreases the absorption of non-haeminic iron absorption.
Thirdly, Yokota et al., (2008) reported that H. pylori strains in iron deficiency anaemia patients showed ferric ion uptake and a fast-iron dependent growth when compared with strains from patients without iron deficiency. The current study therefore hypothesized that the mechanisms revealed by Yokota et al., (2008) could be another possible mechanism used by H. pylori infection in this population to cause anaemia. In the H. pylori positive children in this, 77.1% had anaemia while 22.9% of the infected children did not have anaemia. This shows that certain strains of H. pylori are iron dependent while other strains are non-iron dependent. That could be the main reason why 77.1% had anaemia while 22.9% did not experience the development of anemia. Interference of iron metabolism and iron absorption, affects the haemopoesis process and hence interferes with Hb levels. Iron is an important complement in the development of Hb. This study noted that the H. pylori infection may have contributed to the low Hb levels in children in this population by interfering with iron absorption and iron metabolism.
Eato Brooks, Morgan, Krakowa, (1991) highlighted that urease production which helps the H. pylori bacteria in overcoming the acidic environment of the stomach is one of the mechanisms which affects the iron metabolism and iron absorption. In addition, the absorption of iron is also interfered with by the destruction of the stomach cell membrane by H. pylori infection. Asahi et al., (2003), noted that the binding of VacA to specialized membrane, functional domain have an implication on cell signaling. Tegtmeyer, Zabler, Schmidt, (2009), noted that VacA inhibited the activation of epidermal growth factor receptor (EGFR) and HER2/Neu, and Erk1/2MAP kinase which play an important role for cell elongation and cell scattering. The above mechanism induced by H. pylori infection interferes with cell membrane structure affecting the iron absorption and hence affects the Hb levels.
Our findings agreed with several studies done elsewhere, Baggett Parkinson, Muty, Gold, Gessner, (2006), assessed an association of H. pylori infection and ID among children of the age of 7-11 years and found a great association after age confounding was done. A strong association in children between the age of 9 years and above was noticed. The current study differs with the above study in that after stratification of age groups this study no statistical significance among the age groups was found. In addition the changes in the gastric physiology and its histology in children caused by H. pylori infections were investigated by Baysoy et al., (2004). This study found that the destruction of the gastric physiology and its histology by H. pylori infections affects the serum iron levels. On the same note, a study done in Germany which was a cross-sectional study (n=1806) noticed 17% decrease in serum ferritin concentration in H. pylori IgG positive individuals than in H. pylori IgG negative individuals (Berg Bode, Blettner, Boing, Brenner, 2001). Moreover, in Korean children a positive statistical significance of H. pylori infections and low mean serum ferritin levels (p<0.001) was noticed (Choe, Kim, Hong, 2003). Furthermore, in Australia a study involving women showed high statistical significance of H. pylori infected women and low serum ferritin levels compared with H. pylori negative women even with the same diet intake of iron (Peach, Bath, Farish, 1998). The current and the above study done in Australia were done in children and women respectively. On the same note the current study agrees that there was an association between H. pylori infection and low Hb levels and the Australian study agreeing that H. pylori infection and low serum ferritin levels was statistically significant, meaning that the role of H. pylori infection on Hb levels is felt across the gender group and across all ages.
In a study done in Korean adolescents (n=937), a significant association between H. pylori seropositivity and anaemia was noticed. In addition in a study done by Darvishi, Katayoun, Hussein, Kamyab ,(2015) in Iran found out that the serological investigation for H. pylori infection revealed that 52 (81.3%) patients with IDA and 10 (14.3%) non-anaemic controls were positive for specific IgG H. pylori antibodies and the difference between the two groups were statistically significant (p<0.0001).
Similarly, a study done in Ethiopia by Taye et al., (2015) which upto 6.5 years of H. pylori infected children was associated with higher prevalence of anaemia and reduction of Hb level and red cell indices.
In another study done in Iran, by Darvishi et al., (2015), a case-control study in children under six years, found a strong relationship between H. pylori infection and iron deficiency anaemia. This study strongly agrees with our findings in that both studies uses the same diagnostic method of IgG antibodies and were on the agreement that, there is association between anaemia and seropositivity of H. pylori infection. However, the difference was that Darvishi et al.,(2015) did an investigation in children under 6 years of age while this study focused on children between the ages of 5-15 years. This study proves that the association between H. pylori infection and anaemia is statistically significant in both older and younger children.
However, there are still controversies among various studies regarding the association between H. pylori infection and anaemia. Some authors believe that variation in H. pylori species is one of the possible factors for disagreement with the findings in the literature.A cross-sectional study done among Israel-Arab children found a great association between low serum ferritin and CagA-positive strain (Muhsen et al., 2009).In double-blind randomized trial on non iron deficient in 3-10 years old children in Elpaso Texas reported that eradication of H. pylori infection by CagA negative strains was associated with a larger serum ferritin increase (Cardenas et al., 2011). On contrary, a German study involving a large population based study could not find any association between the low serum ferritin and the seropositivity of H. pylori CagA strains (Berg et al., 2001). On same findings Ciacci et al., (2004) reported a negative association between H. pylori infected adults with CagA positive strains and iron absorption impairment. However, with above controversies, the current study believes that different strains of H. pylori infection associate differently with anaemia. This is due to the fact that 77.1% of H. pylori infected children had anaemia while 22.9% did not develop anaemia, postulating that this could be due to different strains of H. pylori infection. The study concludes that there is astrong association between H. pylori infection and anaemia and recommends that children with low Hb level should be subjected to H. pylori diagnostic tests and treated if found positive.

Conclusion
In conclusion, our findings indicated that seropositivity of H.pylori infection is highly associated with the occurrence of anaemia. Children with unexplained cause of anaemia should be tested for H. pylori infection and treated if found positive.