Is Helicobacter Pylori a Reason for Unexplained Iron Deficiency Anemia: A Systematic Review

Iron deficiency anemia (IDA) is a worldwide public health problem affecting millions, with developing nations accruing a significant disease burden. Helicobacter pylori (H. pylori) has been proposed in many studies as a causative factor for unexplained iron deficiency anemia. In this systematic review, we searched PubMed, Google Scholar, and ScienceDirect to come up with five cross-sectional studies and five Randomized Controlled Trials (RCTs), which evaluated the association between H. pylori and unexplained iron deficiency anemia and the response of IDA to anti-H. pylori therapy. H. pylori eradication therapy included triple therapy (proton pump inhibitor, clarithromycin, amoxicillin) or quadruple therapy (proton pump inhibitor, bismuth, metronidazole, tetracycline) for 10-14 days. Quadruple therapy was used if there is a penicillin allergy or a local antibiotic resistance level of more than 15% to clarithromycin. The cross-sectional studies concluded that H. pylori infection was associated with low serum ferritin levels. The RCTs confirmed that H. pylori are associated with iron deficiency anemia by demonstrating improvement in markers of iron status (ferritin, hemoglobin, Mean Corpuscular Volume (MCV), serum transferrin receptor levels) with H. pylori eradication therapy. In a nutshell, this systematic review concludes that H. pylori testing and treatment must be considered as a differential diagnosis of unexplained IDA in all age groups and serves as a benchmark for more randomized clinical trials to prove causation.


Introduction And Background
Anemia is a global public health problem, mostly in developing nations. World Health Organization (WHO) estimates that over 700 million people worldwide have iron deficiency anemia (IDA) [1]. Poor iron intake, gastrointestinal parasitic infections, low bioavailability of dietary iron, defective iron absorption, and excessive iron loss are the major factors contributing to iron deficiency anemia [2][3]. Iron deficiency in children is associated with deficits in immune, cognitive, and motor function [4]. Unexplained or refractory iron deficiency anemia accounts for about 15% of all IDA [5].
Helicobacter pylori (H. pylori) has been postulated as an etiology for unexplained iron deficiency anemia. The prevalence rate of H. pylori infection is 50% in adults in developed countries and 60%-90% in developing countries [6]. H. pylori-induced chronic gastritis depletes gastric acid secretion and gastric ascorbic acid levels, which are necessary for iron absorption [7][8]. Patients with H. pylori infection show high intragastric pH leading to defective iron absorption and consequent IDA [9]. Other causes of IDA are the sequestration of iron by H. pylori and the loss of iron through the gastrointestinal tract because of inflammatory mucosal injury [8]. Children in developing nations are more likely to develop iron deficiency anemia from H. pylori infection as they may have diminished iron stores due to dietary deficiency [10].
Many studies evaluating the association between H. pylori infection and IDA are cross-sectional. There are also some randomized controlled trials assessing the response of markers of iron status to H. pylori eradication therapy in infected individuals with or without IDA. Some of these studies have been performed on the Alaskan native population because 20%-50% of them continue to have depleted iron stores despite adequate dietary iron and Vitamin C intake [11]. Anti-H. pylori therapy includes standard triple therapy in which a proton pump inhibitor (PPI), amoxicillin, and clarithromycin are given for 10-14 days to eradicate the infection. In case of penicillin allergy, modified triple therapy with replacement of amoxicillin with metronidazole can be given, while PPI and clarithromycin can continue. The treatment regimen for H. pylori depends on local antibiotic susceptibility. In areas of more than 15% macrolide (clarithromycin) resistance, quadruple therapy is initiated, which includes treatment with PPI, bismuth, metronidazole, and tetracycline for 10-14 days. The quadruple regimen can also be used if there is treatment failure on triple therapy. In this systematic review involving global studies, the role of H. pylori as a cause of unexplained IDA and the usefulness of H. pylori eradication therapy in improving IDA or its parameters are explored. 1 1 1 1 1

Search strategy
This study was undertaken per PRISMA (Preferred Reporting Items for Systematic Reviews and Metaanalysis) guidelines [12]. Databases  The search included free full-text articles-observational studies, systematic reviews, or randomized clinical trials conducted on human subjects and published in English from 1995 to 2022. We also used Google Scholar and ScienceDirect to find relevant articles on H. Pylori and iron deficiency anemia. We also used reference lists of original articles and the 'Related Articles' section of PubMed to find relevant articles.

Study selection
Two reviewers independently screened the titles and abstracts of all identified studies for eligibility. The following inclusion criteria were applied: (1) cross-sectional studies, case-control studies, systematic review articles, randomized controlled trials, and meta-analyses (2) all age groups regardless of gender (3) any geographic regions of the world.
Case reports/series, books, letters to the editor, conference abstracts, author's replies, and editorials were excluded. Those articles which required permission for access or payment requirement were also excluded. Articles related to iron deficiency anemia in the pregnant population and anemia of chronic disease were excluded.

Data extraction
Data were extracted independently by two reviewers who initially screened the titles and abstracts for eligibility. A full-text review of the eligible studies was done by two authors independently according to eligibility criteria. The following data were extracted: Study, study design, study population, age of participants, number of participants, the prevalence of H. pylori infection, classification of Iron Deficiency (ID)/ Anemia/Iron Deficiency Anemia (IDA), and H. pylori detection method.
For cross-sectional studies, we extracted the data on the H. pylori infection status of study participants based on Immunoglobulin G (IgG) antibody levels/urea breath test/stool antigen test/endoscopy, the prevalence of H. pylori infection in percentage, the measures of association between H. pylori infection and iron deficiency anemia using Odds Ratio (OR) or relative risk (RR). For randomized clinical trials, data were collected regarding the number of participants in the intervention and control group, the treatment received by each group, and the baseline and follow-up levels of markers of iron status, including serum ferritin (SF), hemoglobin (Hb), Mean Corpuscular Volume (MCV) and serum transferrin receptor (sTfR) levels. Any significant association between H. pylori infection and the markers of iron status, which demonstrated a pvalue of less than 0.05, was considered to interpret the results of this systematic review.

Methodological quality assessment
The risk of bias in studies included in this systematic review was assessed independently by two reviewers, and disagreements were resolved by consensus. Quality assessment of cross-sectional studies was done using the Adapted Newcastle Ottawa Quality Assessment Scale developed by Herzog et al. [13]. The scale included three main categories: selection, comparability, and outcome. A maximum of one point is given for each numbered item in the selection and exposure category, and a maximum of two points is given for comparability. This contributes to a maximum score of four, two, and two, respectively, in the selection, comparability, and outcome categories of the Adapted Newcastle Ottawa Quality Assessment Scale. The Cochrane Collaboration's tool was used for the quality assessment of randomized clinical trials included in this review [14]. The bias was assessed as high, low, or unclear for seven individual elements spread over five domains (selection, performance, attrition, reporting, and others).

Literature search
A flow diagram of study identification and subsequent inclusion is shown in Figure 1. A total of 7912 citations were identified by the initial search, in which 10 studies were selected for this systematic review after the screening, eligibility criteria, and quality assessment. A total of 7144 patients were included in this study.

Characteristics of the included studies
The characteristics of the included studies are shown in Table 1. These studies spanned from 1998 to 2021 and were done in different parts of the world. The number of participants varied from a minimum of 86 to a maximum participant number of 2794. Both children and adults were included in our study. Five studies evaluated participants' responses to H. pylori eradication therapy concerning iron deficiency anemia. Four studies among them were randomized controlled trials [6,9,[15][16], and one was an uncontrolled trial [17]. Five out of ten studies chosen for systematic review were cross-sectional studies [4,[7][8]10,18]. Among the studies, three were conducted in Alaska [8,15,18], three were conducted in Asia [6][7]9], and others in Europe [10], the United States [16], and Egypt [17]. The study by Queiroz et al. [4] was an international multicenter trial conducted in South American countries (Chile, Brazil) and Europe (UK).   Table 2 demonstrates the risk of bias assessment for randomized controlled trials done by the Cochrane Collaboration's tool [14]. Among the RCT, three studies depicted a low risk of bias [6,[15][16], and two studies [9,17] had an unclear risk of bias with low risk in at least two domains.   Table 3 demonstrates the risk of bias assessment for cross-sectional studies using the Adapted Newcastle Ottawa scale [13]. Three studies were classified as good, with a total score of 7-8 [4,8,10]. Two studies were satisfactory, scoring 5-6 [7,18].  mg/L, p = 0.02) had lower median serum ferritin levels compared to respective IgG negative category. This difference was not significant in pre-menopausal women (p = 0.13). The study also found that H. pylori seropositive postmenopausal women were more likely to have iron deficiency (serum ferritin < 15 mg/L) compared to seronegative postmenopausal women (p = 0.03).

Random
Another population-based serosurvey by Parkinson

Trials evaluating response to H. pylori eradication therapy influencing iron-related parameters
Cardenas et al. conducted a double-blind, randomized controlled trial in El Paso County, Texas, between 2006 and 2008 in 110 H. pylori-infected children without iron deficiency and between 3 to 10 years [16]. The intervention consisted of quadruple sequential treatment of lansoprazole and amoxicillin for the first five days, followed by lansoprazole + clarithromycin + tinidazole for the next five days. Ferrous sulfate supplementation consisted of 3 mg/kg/day of elemental iron for six weeks. Four treatment arms were defined: quadruple eradication + iron supplementation, quadruple eradication only, iron supplementation only, and placebo. In the intent to treat (ITT) analysis, subjects received at least one dose of H. pylori eradication treatment. As per ITT analysis, only 44% of children receiving H. pylori eradication alone or combined with iron cleared their infection. Per protocol (PP) analysis accounted for losses to follow-up and included 90 children. In both ITT (n = 110) and PP (n = 90) analysis, there were no significant differences across study arms on means and mean differences of hemoglobin, serum ferritin, or transferrin saturation from baseline levels at six months follow-up. However, the children who were eradicated from H. pylori at 45 days showed a statistically significant larger increase in mean ferritin levels (7.7 ng/mL, p <0.05) than children who remained infected (1.9 ng/mL). The study also showed that eradicating infection by CagA (cytotoxin associated gene product A) negative strain showed a larger increase in ferritin than CagA positive strain. pylori-positive compared to 28.1% (235 of 835) in the non-anemic group, and this difference was statistically significant (p <0.01). In multiple logistic regression analysis, a large volume of menstruation (r 0·239, OR = 1·270, 95 % CI 1·059, 1·523) and H. pylori infection (r 0·347, OR = 1·360, 95 % CI 1·212, 1·638) were found as risk factors for anemia. Eighty adolescent girls with IDA and H. pylori infection were randomized into the intervention group (oral iron + triple therapy, n = 32) and control group (oral iron only, n = 42) after exclusion criteria. It was found that the mean values of Hb and SF increased and sTfR decreased after treatment compared with those before treatment in the intervention group (p <0.01). This difference was maintained in the H. pylori-negative group (n = 31, p <0.01) after treatment compared to before therapy after re-division based on H. pylori eradication status following the intervention trial.
Chen et al. conducted a randomized controlled trial in 86 adult patients with iron deficiency anemia and H. pylori-associated chronic gastritis. They evaluated the effect of H. pylori eradication on the response to oral iron therapy by dividing them into two equal-sized groups of 43 each [9]. Group A cases received both oral iron treatment and H. pylori eradication therapy, while Group B only received oral iron treatment.

Discussion
This systematic review found an association between H. pylori infection and iron deficiency anemia. Two forms of iron are absorbed in the duodenum and jejunum part of the gastrointestinal tract: heme iron and non-heme iron. Heme iron is mainly present in meat and is easily absorbed [7]. Non-heme iron is primarily present in vegetables and grains and requires conversion from the oxidized ferric form to the ferrous form to get absorbed [10]. The reduction of ferric to the ferrous form of iron is facilitated by acidic gastric pH and ascorbic acid. H. pylori increase gastric pH by causing atrophic gastritis, uses iron for its growth and acts as a causative factor for iron deficiency anemia. The outer membrane proteins in H. pylori bind ferritin and lactoferrin with high affinity [19][20]. H. pylori can also alter iron metabolism by iron sequestration [21]. Occult gastrointestinal bleeding caused by H. pylori-induced peptic ulcer disease is a less common cause of iron deficiency anemia than the causes mentioned above. It has been proposed by the Maastricht V/Florence Consensus Report that patients with unexplained iron deficiency anemia must be tested and treated for H. pylori infection. However, the grade of recommendation is weak [22]. (MH test), p = 0.013) and these studies were adequately powered with a sample size of more than 2000 study subjects [10,18]. Queiroz et al., in their multicenter study, found an association between H. pylori infection and low serum ferritin and hemoglobin in a combined analysis of the population of Chile and Brazil, which were nations with similar socio-economic development [4].

Association between H. pylori infection and iron deficiency anemia
All the randomized clinical trials included in this systematic review concluded that H. pylori eradication therapy increased serum ferritin levels from baseline. While Cardenas et al. [16] and Fagan et al. [15] did not report an increase in mean hemoglobin levels in the H. pylori eradicated patients, other studies [6,9,17] demonstrated that there was an increase in hemoglobin levels in subjects who became H. pylori-negative upon treatment.
Three cross-sectional studies included in this review reported that the seroprevalence of H. pylori in terms of IgG positivity increased with age [8,10,18]. This was identical to the results obtained by Lim [24]. A similar trend is seen in developing nations and may be attributed to the socio-economic status, crowded living places, and the unavailability of piped water supply [24].

H. pylori infection and its influence on serum ferritin levels
All the studies included in this review concluded a correlation between H. pylori infection and low serum ferritin levels. In a large-scale survey of 7462 participants, H. pylori infection was associated with a 13.9% decrease in serum ferritin levels and with the prevalence of IDA (OR 2.6, CI 1.5 -4.6) [25]. A cross-sectional study conducted by Berg et al. in Germany also reported a 17% decrease in serum ferritin (CI 9.8 -23.6) in the H. pylori-infected group in multiple regression analysis in a sample size of 1806 individuals. Berg et al. also found a similar decrease in serum ferritin levels in infection with both Cag-A positive and negative strains [19]. This is contrary to the findings of Cardenas et al., who found a significant increase in ferritin levels with the eradication of infection in the Cag-A negative strain [16].
The increase in serum ferritin level after H. pylori eradication therapy, as depicted by the clinical trials included in this review, is endorsed by the studies of Choe et al. [26]. Metanalysis by Qu et al. suggested that IDA is associated with H. pylori (pooled OR 2.22, 95% CI 1.52 -3.24 (p < 0.0001), and the eradication of H. pylori improves IDA [27]. These conclusions were contradicted by the studies of Gessner et al., who documented that the resolution of H. pylori infection did not improve iron deficiency or anemia up to 14 months after treatment initiation [28]. A possible explanation is that atrophic gastritis caused by H. pylori infection leading to iron deficiency anemia may take more than 14 months for resolution. Tseng et al. did not find significant improvement in IDA after H. pylori treatment when followed up for up to two years [29].
A retrospective cross-sectional study in older adults did not demonstrate an association between iron deficiency anemia and H. pylori infection [30].
Since all of the trials show an improvement in ferritin levels with therapy, our analysis stresses that ferritin is the first marker of iron status to improve with the removal of H. pylori and iron supplementation. The most effective test for iron deficiency is serum ferritin, which indicates the body's mobilizable iron reserves [10]. Our study emphasizes the relevance of implementing a national guideline for treating refractory iron deficiency anemia with H. pylori eradication therapy. This review reflects on the necessity to address the burden of unexplained anemia in the community by addressing the impact of the highly prevalent H. pylori infection.

Limitations
One of the limitations of our study was the cross-sectional design of five of the included studies. Also, we could not demonstrate an improvement in iron deficiency anemia in some of the clinical trials included in the analysis. However, we were able to emphasize an improvement in serum ferritin and hemoglobin levels with anti-H. pylori therapy. Also, we could not tailor our study to specific populations with a high incidence of iron deficiency anemia, namely pregnant and pre-menopausal women. Also, the increase in serum ferritin seen in anemia of chronic disease could not be accounted for in our study. However, this systematic review includes large-scale cross-sectional and randomized trials, which prove the association of iron deficiency anemia with H. pylori infection and serves as a benchmark for future clinical trials.
following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.