Complete Blood Counts among chronic patients of Helicobacter pylori infection

Introduction

Helicobacter pylori (H. pylori) is a gram-negative micro-aerophilic bacterium, considered the most common chronic infection affecting humans worldwide. [1] Approximately 4.4 billion individuals were infected globally with H. pylori during 2015 with prevalence from 8.7% to 85.5%, increasing with age and with large variations regarding race, ethnicity, socio-economic status, and geographical area, over 80% of them being in developing countries [2-7]. Recently many researchers reported H. pylori to be the principal cause of chronic gastritis and the main etiological agent for anaemia, gastric cancer, peptic ulcer disease, ischemic cerebrovascular disease, mucosa-associated lymphoid tissue (MALT), lymphoma, dermatologic, neurologic disorders, metabolic and cardiopulmonary diseases. [2,8-12] Furthermore, previous studies reported association of H. pylori infection with many haematological manifestations, such as vitamin B12 deficiency, iron deficiency anemia (IDA) and Idiopathic Thrombocytopenic Purpura (ITP). [13-14] The frequency of anaemia is high in many countries infected by H. pylori bacteria, schistosomiasis and malaria, particularly in developing countries, causing serious public health problems. [15, 16-18] Given the high prevalence of H. pylori infection reported in Sudan, as the commonest etiological factor of gastritis, [19] our current study rationale is to explore and establish the effect of these bacteria on the haematological parameters in Sudanese H. pylori-infected patients in order to help in the management of H. pylori, which is highly recommended for high-risk populations [8, 20].

Methodology

Study design and study setting: A case-control study was done at National Health Insurance Hospital, Kosti, Sudan from May to October 2018.

Participants and sample size: In accordance with exclusion and inclusion criteria, one hundred and thirty participants were randomly recruited in the current study. Eighty participants had H. pylori infection (16 males, 64 females), aged 16-55 years, of mean age (32.95±10.39); As control, we selected 50 healthy persons matched in age and gender without gastrointestinal tract infection, and with negative H. pylori laboratory tests, who attended the National Health Insurance Hospital and agreed to participate.

Data collection: Data was collected using structural interviewing questionnaire, which included personal and clinical information, and laboratory investigations. Three ml of venous blood were taken and collected in sterile dry plastic syringes using standard venipuncture technique from each participant after sterilizing the puncture site with 70% ethanol.

The samples were collected in Plain containers for detection of antibodies against H. pylori organisms, the former centrifuged at 2000 rpm (revolutions per minutes) for 15 minutes, then the supernatant sera used, without preservation or delay using ICT, which utilizes a combination of Helicobacter pylori antigen, coated particles and anti- human IgG to selectively and qualitatively detect H. pylori antibody in the serum.

The blood samples collected in Ethylene diamine tetra acetic acid (EDTA) containers were used for Complete Blood Counts (CBC), which was done using (Sysmex, Tokyo, Japan-XK-21SYSMEX) haematology-automated analyser. Peripheral Blood Picture (PBP) were done using Romanowsky stains.

Stool samples were collected in clean, dry containers, which were examined immediately by mixing small amounts of stool, and one drop of this mixture was taken for ICT shaken and incubated for 1 minute and then read.

Inclusion and Exclusion Criteria: All included participants reported symptoms of gastritis and had laboratory-confirmed diagnosis of H. Pylori infection, and attended Kosti National Health Insurance Hospital. Patients were excluded from participation if they had been diagnosed with blood diseases, cardiovascular disease, or any type of haemorrhagic disease. Also excluded were patients taking medication affecting blood parameters, those with gastric malignancy or malignant diseases, and those with other causes of thrombocytopenia. Finally, some patients refused to participate also excluded.

Statistical analysis: Laboratory results were analyzed, by IBM SPSS (Statistical Package for the Social Sciences) version 25. Descriptive statistics and frequency distribution was performed. Mean (M) ± standard deviation (±SD) calculated, and comparison was done with two-tailed Student's t-test to explore any significant difference between H. Pylori infected patients and healthy individuals’ control group. Alpha error of 0.05 and P value ≤0.05 was considered as significant.

Informed consent: Prior to collecting the blood samples, each participant completed the informed consent form. Parents completed and signed informed consent form for patients below 18 years old.

Ethical approval: This was obtained from the research and ethics committee of El Imam El Mahdi University, Faculty of Medical Laboratory Sciences.

Results: 80% of the current study participants were female, 20% were male amongst both the case and control groups, 40% and 46% of the patients and controls respectively were aged between 25-34 years old. 28.7% and 26% % were between 35-44 years old, 23.8% and 20% were 45-55 years old, while 7.5% and 8% were aged between 15-24 years old. The mean age for the H. Pylori infection group was 32.95±10.39 years old and 31.36±8.75 for the control group, with no statistically significant difference between the two groups (p value = 0.900) (Table 1). The mean of red blood cell counts (RBCs), haemoglobin (Hb), Haematocrit (Hct), mean cell volume (MCV), neutrophil and lymphocyte were 4.2×1012/cmm (± 0.7) vs. 4.8×1012/cmm (±0.3); 12.0 g/dl (±1.8) vs. 13.4 g/dl (±1.4); 34.2 l/l (±6.1) vs. 37.2 l/l (±4.2); 83.1 fl (±6.7) vs. 86.6 fl (±4.9); and 49.6% (±12.2) vs. 53.6% (±7.7) and 41.9% (±11.1) vs. 35.7% (±7.3) in cases with H. Pylori and controls respectively. Our findings show that some haematological parameters, RBCs, Hb, Hct, MCV and neutrophil were significantly decreased among the case group compared to the control group, (p values = 0.001, 0.001, 0.003, 0.002 and 0.001 respectively) with the exception of lymphocyte count which was significantly elevated in H. Pylori infected patients over controls, with p value <0.01, as shown in Table 4,5. No significant differences between our study groups were observed in the following parameters: TWBCs, platelet, monocyte, and eosinophil counts. They were 6.0 ×103/l (±2.1) vs. 6.4 ×103/l (±1.9); 284.9×103/l (±77.3) vs. 292.4 ×103/l (±69.4); monocyte 5.4 % (±3.0) vs. 6.2 (±2.7)%; eosinophil 3.4 (±2.2)% vs. 4.7 (±2.4)%; HCH 28.8 pg (±7.3) vs. 30.4 pg (±2.2) and HCHC 34.4% (±2.5) vs. 34.9% (±1.9) respectively, as shown in Tables 4,5. Peripheral blood picture of most H. Pylori infection patients was microcytic hypochromic RBCs. Macro-ovalocytes, anisocytosis, poikilocytosis with teardrop; target red cells and Echinocyte were observed. However, normocytic and normochromic picture was observed in some patients with H. Pylori infection. White blood cell morphology of H. Pylori infection patients showed lymphocytosis and neutropenia.

Table 1. H. Pylori infected patients and control group age and gender
P value	t	Control group N=50	H. Pylori infected N=80	Category	Parameter
	Frequency (%)	Frequency (%)
		10 (20)	16 (20)	Male	Gender
		40 (80)	64 (18)	Female
50 (100)	80 (100)	Total
		4 (8)	6 (7.5)	15-24 years old	Age distribution
23 (46)	32 (40)	25-34 years old
10 (20)	19 (23.8)	35-44 years old
13 (26)	23 (28.7)	45-55 years old
0.37	0.90	31.36±8.75 years old	32.95±10.39 years old	Mean ± SD

Table 4. Results of red cell indices among H. Pylori infected patients against control samples
Parameter	Patient M (±SD)(N=80)	Control M(±SD)(N=50)	P-value
Hb g/dl	12.04 (±1.84)	13.4 (±1.4)	<0.001*
PCV l/l	34.2 (±6.1)	37.2 (±4.2)	<0.003*
RBCs × 106/ l	4.2 (±0.7)	4.8 (±0.3)	<0.001*
MCV fl	83.1 (±6.7)	86.6 (±4.9)	<0.002*
MCH pg	28.8 (±7.3)	30.4 (±2.2)	0.062
MCHC%	34. (±2.5)	34.9 (±1.9)	<0.185

SD= Standard Deviation

Table 2. Red blood cell indices of the participants
Variable	H. Pylori infected patients (%)N=80	Control group N (%)N=50
Hb
<13.5 g/dl	56 (70%)	29(58%)
13.5-17.5 g/dl	24(30%)	21(42%)
>17.5 g/dl	0(0%)	0(0%)
PCV
<40 l/l	71(88.7%)	40(80%)
40-52 l/l	8 (10%)	10 (20%)
>52 l/l	1(1.3%)	0(0%)
RBCs
<4.5 x 1012	56 (70%)	1 (2%)
4.5-6.5 x 1012	23(28.7%)	49(98%)
>6.5 x 1012	1(1.3%)	0(0%)
MCV
<80 fl	22(27.4%)	4(8%)
80-95 fl	53(66.3%)	45(90%)
>95 fl	5(6.3%)	1(2%)
MCH
<27 pg	17(21.3%)	2(4%)
27-34 pg	60 (75%)	48(96%)
>34 pg	3(3.7%)	0(0%)
MCHC
<20%	0(0%)	0(0%)
20-35%	52(65%)	30 (60%)
>35%	28(35%)	20(40%)

Key: Hb = Haemoglobin concentration; PCV= Blood Cell Volume; RBCs = Red Blood Cells Count; MCV= Mean Cell Volume; MCH = Mean Cell Haemoglobin; MCHC= Mean Cell Haemoglobin Concentration.

Table 3: TWBC, PLT and Differential White Blood Cell Counts group.
Variable	H. Pylori infected patients (%)N=80	Control group N (%)N=50
TWBC
<4×103 /l	9(11.3%)	2(4%)
4-11×103 /l	68(85%)	47(94 %)
>11×103 /l	3(3.7%)	1(2%)
PLt
<150×103 /l	1 (1.3%)	0(0%)
150-400×103 /l	70(87.4%)	45(90%)
>400×103 /l	9(11.3%)	5(10%)
Neutrophil
<40%	16(20%)	2(4%)
40-80%	2(2.5%)	2(4%)
>80%	62(77.5%)	46(92%)
Lymphocyte
<20%	1(1.3%)	0(0%)
20-40%	35(43.7%)	44(88%)
>40%	44(55%)	6(12%)
Monocyte
<4%	10(12.5%)	6(12%)
4-10%	70(87.5%)	42(84%)
>10%	0(0%)	2(4%)
Eosinophil
1%	18(22.5%)	2 (4%)
2-4%	41(51.3%)	26(52%)
>4%	21( 26.2)	22(44%)

Key: TWBC = Total White Blood Cells Counts; PLt = Platelets count

Key: - M=Mean; *Significance at the level ≤0.05

Table 5: Results of TWBC, PLT and Differential White Blood Cell Counts among H. Pylori infected patients against control samples
Parameter	Patient M (±SD)(n=80)	Control M(±SD)(n=50)	P-value
TWBC ×103/l	6.0 (±2.09)	6.4 (±1.9)	0.818
PLt×103/l	284.9 (±77.3)	292.4 (±69.4)	0.556
Neutrophil%	49.56±12.23%	53.6 (±7.8)	<0.001*
Lymphocyte%	41.9 (±11.1)	35.7 (±7.3)	<0.004*
Eosinophil%	3.4 (±2.2)	4.7 (±2.4)	0.673
Monocyte%	5.4 (±3.0)	6.2 (±2.7)	0.113

Key: - M=Mean; *Significance at the level ≤0.05

Discussion

H. pylori are closely related to haematological disorders such as iron deficiency anaemia (IDA), and primary immune thrombocytopenia (ITP). [11] The current study aims to detect the effects of H. Pylori infection on haematological parameters among Sudanese patients attending Kosti health insurance hospital and compare their results with those of healthy individuals. Our findings were in accordance with recent studies which were conducted in other ethnic groups [21, 22] and another study done in Sudan, [12] which stated decrease in some haematological parameters (Hb, Hct, MCV and neutrophil counts) in H. pylori patients. Our findings were however not in agreement with the results of the study done in Saudi by Nahla KI and co-workers who found no association between H. pylori and haematological parameters. [23] Furthermore, a retrospective study was conducted between 2012 to 2016 in China by MeiYX et al [24] , who found that, H. pylori infection may reduce the level of Hb and in turn may be responsible for causing anaemia in a Chinese population. In contrast previous studies were carried out in a Nigerian tertiary educational institution by Benjamin OE and co-workers [25] who mentioned a non-significant difference in MCV values and another study done among Palestinian adult patients infected with H pylori by Saleh NM and Wesam MA [26] who concluded similar results regarding MCV which was not the same as that found here. Our results are in agreement with the previous study done by Waleed S, et al [27] , in 2016 in Taif City, Saudi Arabia and the study done by Saleh NM and Wesam MA [26] , while inconsistent with the study by Ashraf AT, et al [28], in 2015 in Egypt who reported insignificant difference in RBCs counts. Our results are consistent with the previous study done in Palestine by Saleh NM and co-workers [26], who reported low level of both MCH and MCHC in patients with H. pylori infection [12, 27]. On the other hand our current findings are consistent with the study done by Elham AI, et al [12] in Sudan, and inconsistent with other studies who concluded high total WBCs count in patients with H. pylori infection patients compared to controls [26, 27]. Concerning Platelet counts, our results revealed no statistically significant difference between H. Pylori infection patients and control group, this finding was in line with other studies [26, 28]. Regarding the Neutrophil counts and the lymphocyte counts our findings were with other studies [29, 30] , which indicated that H. pylori infection may be related to anaemia with high neutrophil and low lymphocyte counts. The significant increase of lymphocytes count may be due to inflammations caused by H. pylori infection. The possible reasons for the differences and similarities in blood parameters with other mentioned studies may include the degree of severity of H. pylori infection, the demographic data such as age of the participants, and the sample size. Our findings concerning the peripheral blood film morphological findings, which are highly variable consistent with earlier studies [12, 31] who reported oval shaped, tear drops, microcytic hypochromic picture among H. Pylori infection patients.

Conclusion

From this study, we conclude that some haematological parameters, particularly Hb, Hct, MCV and neutrophil counts were decreased among patients with H pylori infection, with the exception of lymphocyte counts, which was high in these patients. Therefore, H. Pylori infection is a cause of anaemia and must be considered while investigating and managing anaemic patients (especially with acid-peptic disease).

Funding: None.

Conflict of Interest: The authors declared no conflict of interests.

Acknowledgement: The authors thank all participants in this study.

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