Acta Clinica Belgica International Journal of Clinical and Laboratory Medicine

ISSN: 1784-3286 (Print) 2295-3337 (Online) Journal homepage: http://www.tandfonline.com/loi/yacb20

Correlation of interleukin-4 levels with Plasmodium falciparum malaria parasitaemia in Sudanese children A. B. Elhussein, M. A. Huneif, A. Naeem, O. E. Fadlelseed, W. G. Babiker, N. E. A. A. Rahma, S. A. M. Ahmed, I. A. M. Ayed & M. H. F. Shalayel To cite this article: A. B. Elhussein, M. A. Huneif, A. Naeem, O. E. Fadlelseed, W. G. Babiker, N. E. A. A. Rahma, S. A. M. Ahmed, I. A. M. Ayed & M. H. F. Shalayel (2015) Correlation of interleukin-4 levels with Plasmodium falciparum malaria parasitaemia in Sudanese children, Acta Clinica Belgica, 70:6, 414-418 To link to this article: http://dx.doi.org/10.1179/2295333715Y.0000000042

Published online: 02 Jul 2015.

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Date: 07 February 2016, At: 22:58

Original Paper

Correlation of interleukin-4 levels with Plasmodium falciparum malaria parasitaemia in Sudanese children A. B. Elhussein1, M. A. Huneif 2, A. Naeem 3, O. E. Fadlelseed1 , W. G. Babiker 4, N. E. A. A. Rahma 5, S. A. M. Ahmed 6, I. A. M. Ayed7, M. H. F. Shalayel8 Department of Biochemistry, Najran University, Saudi Arabia, 2Department of Pediatrics, College of Medicine, Najran University, Saudi Arabia, 3Department of Biochemistry, Omdurman Islamic University, Sudan, 4Najran University, Saudi Arabia, 5Department of Pediatrics, University of Bahri, Sudan, 6Nile College, Sudan, 7Faculty of Medicine and Health Sciences, Omdurman Islamic University, Sudan, 8College of Medicine, Najran University, Saudi Arabia

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Objectives: This study aimed to measure the level of interleukin-4 (IL-4) in the serum of children patients with falciparum malaria and to correlate the production of this cytokine with the severity of malaria parasitaemia. Methods: One hundred ten patients with malaria participated in this study (53 males and 57 females) and their results were compared with that of 60 healthy control subjects. Their ages ranged between 6 months and 15 years. For the detection of parasitaemia, a calibrated thick-smear technique was used with standard Giemsa staining. For designation of the relative parasite count, a simple code from one to four crosses is used according to the criteria mentioned by Gilles and Warrell. The blood samples were assessed for IL-4 using enzyme-linked immunosorbent assay (ELISA) technique. Results: Thirty-three malaria patients (30.27%) had one cross (z) parasitaemia, 13 patients (11.93%) had (zz) parasitaemia, 24 patients (22.02%) had (zzz) parasitaemia and 39 patients (35.78%) had (zzzz) parasitaemia. There was a significant difference (Pv0.009) in the concentration of IL-4 between malaria patients (160.74+ 25.5 pg/ml) and control group (62.136 + 18.16 pg/ml). Uncomplicated malaria patients showed the highest record of IL-4 level followed by cerebral malaria (CM) group and then severe malaria anaemia group (SMA) (255.8+ 54.13, 102.7 + 34.88 and 90.95+ 20.90 pg/ml respectively, Pw0.0001). Conclusion: It was concluded that elevation of serum IL-4 in Sudanese children suffering from Plasmodium falciparum malaria is correlated with the severity of malaria hyperparasitaemia rather than with the severity of the disease. Keywords: Plasmodium falciparum, Cerebral malaria (CM), Parasitaemia, Interleukin-4 (IL-4)

Introduction Malaria is a major health problem in the tropical and temperate regions of the world, which poses a significant burden on health expenditure.1,2 It is caused by obligate intra-erythrocytic protozoa of the genus Plasmodium. Humans can be infected with one (or more) of the following five species: Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae and Plasmodium knowlesi. Plasmodia are primarily transmitted by the bite of an infected female Anopheles mosquito, but infections can also occur through the

Correspondence to: Mohammed H. F. Shalayel, College of Medicine, Najran University, Saudi Arabia. Email: [email protected]

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DOI 10.1179/2295333715Y.0000000042

exposure to infected blood products (transfusion malaria) and by congenital transmission. Among the five species, P. falciparum is the predominant one in Sudan, and is responsible for most of the malariarelated morbidity and mortality.3 Malaria is the most important parasitic disease of human beings. It is transmitted in 108 countries inhabited by roughly three billion people, and, in 2010, caused an estimated 216 million cases and 655 000 deaths.4 More than 85% of malaria cases and 90% of malaria deaths occur in sub-Saharan Africa, mainly in young children (i.e. those younger than 5 years). Malaria is a protozoan disease transmitted by Anopheles mosquitoes. Five species of the genus Plasmodium

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cause all malarial infections in human beings. Most cases are caused by either P. falciparum or P. vivax, but human infections can also be caused by P. ovale, P. malariae, and, in parts of Southeast Asia, the monkey malaria P. knowlesi.5 Infection with P. falciparum displays a remarkable range of disease severity from asymptomatic to severe disease. In severe malaria, certain cytokine levels are increased that have been implicated in pathophysiology of malaria.6 The regulation of antibody profiles in patients with complicated and uncomplicated malaria is still largely unknown. In both human and mice, different cytokines are thought to induce particular Ig isotypes. In humans, interleukin-4 (IL-4) regulates B cells to express the c1, c2, c3, c4, a1, a2 and e, germline gene transcripts (GLT) and to secrete the corresponding proteins.7 The IL-4 is a cytokine that induces differentiation of naive helper T cells (Th0 cells) to Th2 cells. Upon activation by IL-4, Th2 cells subsequently produce additional IL-4 in a positive feedback loop. The cell that initially produces IL-4, thus inducing Th0 differentiation, has not been identified, but recent studies suggest that basophils may be the effector cell.8 The main aim of this study was to measure the level of IL-4 in the serum of children patients with falciparum malaria and to correlate the production of this cytokine with the severity of malaria parasitaemia.

Patients and Methodology One hundred ten patients with malaria participated in this study (53 males and 57 females) and their ages ranged between 6 months and 15 years. They were admitted to Sennar Teaching Hospital (Sudan). The patients were confirmed to have malaria through blood-film examination and 60 healthy children from Sennar area were randomly selected as control group. Classification of each case was based on symptoms, physical signs and laboratory results of malaria at the time of first presentation. P. falciparum malaria was established by the microscopic diagnosis of P. falciparum parasites in the peripheral blood and clinical signs according to the World Health Organisation (WHO) criteria. For the detection of parasitaemia, a calibrated thick-smear technique was used with standard Giemsa staining. According to haematological parameters measured, parasitaemia and neurological evidences, the severity of disease was divided into three groups: uncomplicated malaria, severe malaria anaemia (SMA) and cerebral malaria. For the designation of relative parasite count, a simple code from one to four crosses is used according to the criteria mentioned by Gilles and Warrell.9 (z) for 1–10

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parasites per 100 thick film fields, (zz) for 11– 100 parasites per 100 thick film fields, (zzz) for 1–10 parasites per one thick film field and (zzzz) for more than 10 parasites per one thick film field. The blood samples were collected for IL-4 assessment in sterile tubes containing EDTA. All the samples were centrifuged and serum was refrigerated at {40uuC using enzyme-linked immunosorbent assay (ELISA) obtained commercially (Sunlong Biotech CO., Ltd). Blood haemoglobin was measured by cyanmethemoglobin method. Severe malaria anaemia was defined according to WHO, which defines it as Hb concentrations v5.0 g/dl (or a haematocrit v15.0%) in the presence of any density parasitaemia.10 The assays were performed according to the manufacturer’s protocol. Each plate included a standard curve and known positive and negative controls. Absorbance was read against a blank at 450 nm using a microtitre ELISA reader. All patients were assured that all their obtained information will be handled in a confidential atmosphere and it will not affect their life after taking verbal and written consent. All the human studies were carried out according to the guidelines of the Animal and Human Ethical Committee of Omdurman Islamic University.

Statistical analysis Results of this study were statistically analysed using (SPSS) programme. Significant differences between groups were assessed by one-way ANOVA and t-test. Correlation matrix was done and the r values were obtained with the level of significance.

Result Our study included 110 malaria patients admitted to Sennar Teaching Hospital infected with P. falciparum. Forty-three patients (39.09%) were diagnosed as severe malaria anaemic patients, 22 patients as cerebral malaria (20%) and 45 patients as uncomplicated malaria (40.91%) in addition to 60 healthy control persons. Thirty-three malaria patients (30.27%) had one cross (z) parasitaemia, 13 patients (11.93%) had (zz) parasitaemia, 24 patients (22.02%) had (zzz) parasitaemia and 39 patients (35.78%) had (zzzz) parasitaemia (Table 1). Our results revealed that there was a significant difference (Pv0.009) in the concentration of IL-4 between malaria patients (160.74+ 25.5 pg/ml) and control group (62.136 + 18.16 pg/ml). Uncomplicated malaria patients showed the highest record of IL-4 level followed by cerebral malaria (CM) group and then SMA group (255.8+ 54.13, 102.7+ 34.88 and 90.95 + 20.90 pg/ml, respectively, Pw0.0001). Levels of IL-4 in different classes of malaria parasitaemia are shown in Table 2 and Fig. 1.

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Table 1 Parasitaemia in the different studied groups Studied groups

Severe malaria anaemic

Parasitaemia Number of patients

þ 12

þþ 7

þ þþ 5

þ þþ þ 19

Cerebral malaria þ 5

þþ 3

þ þþ 3

Uncomplicated malaria

þ þþ þ 11

þ 16

þþ 4

þ þþ 16

þ þþ þ 9

Table 2 Levels of interleukin-4 in malaria with different classes of parasitaemia and control group Groups

(þ ) Parasitaemia

(þ þ) Parasitaemia

(þ þþ ) Parasitaemia

(þþ þþ ) Parasitaemia

Control

Interleukin-4 pg/ml 95% CI

104.63 ^ 36.46*

142.05 ^ 73.17**

166.06 ^ 56.00***

211.65 ^ 48.39#

62.136 ^ 18.19

31.25–53.74

59.10–100.73

87.86–119.99

135.86–163.17

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Data are expressed as Mean ^ SD. *P,0.05 vs control group; **P,0.01 vs control group; ***P,0.001 vs control group; # P,0.0001 vs control group.

Figure 1 Levels of interleukin-4 in different classes of malaria parasitaemia.

Pearson correlation coefficient of IL-4 with malaria parasitaemia was {0.320. Similarly, Spearman’s rho correlation coefficient of IL-4 with malaria parasitaemia was {0.360. This indicates that correlation is significant (two-tailed) at the 0.01 level.

Discussion Complications of severe anaemia and cerebral malaria are thought to be the major cause of morbidity and mortality but some evidence suggests that the host’s immunological response could also contribute to the pathophysiology of the disease.11,12 Intensive studies of the immune response to malaria parasites in human beings have provided a wealth of information about the cells and cytokines implicated in the pathophysiology of survival and fatal outcome in severe infections.13 Our results revealed that the concentration of IL-4 is significantly higher in malaria patients than in normal control group and it significantly increases with

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increasing parasitaemia. P. falciparum infection causes an increase in blood concentration of both pro-inflammatory cytokines, such as TNF-alpha, IL-1, IL-6, IL-18, and anti-inflammatory TH-2 cytokines, IL-4 and IL-10. The balance between these inflammatory mediators is critical to parasite control. However, their role in pathogenesis of disease remains unclear.14,15 Other evidences suggested that malaria outcome depends on cytokine overproduction and not on the balance between them, since high levels of anti-inflammatory as well as pro-inflammatory cytokines may be associated with disease severity and mortality.16,17 Anti-inflammatory cytokines (like IL-4) are typically produced during the later stages of the innate immune response to P. falciparum in which they downregulate the potentially pathogenic pro-inflammatory responses that are important for controlling parasitaemia.10 Interleukin-4 interferes with TH-1 cell development

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and reduces the production of IFN-gamma. It inhibits macrophage-mediated killing of P. falciparum. Interleukin-4 impairs an effective antibody response in two ways: by the inhibition of interferon gamma production, it reduces the activity of the cytokine, which mediates immunoglobulin subclass switching to the opsonising IgG1 and IgG3 sub-classes, which mediate parasite phagocytosis by activated monocytes. Furthermore, IL-4 inhibits Fc-gamma receptor expression on phagocytes. The downregulation of immunoglobulin receptors might explain the higher immunoglobulin levels in patients with cerebral malaria.18 In our results, levels of IL-4 in cerebral malaria group and severe malaria anaemic group were significantly higher than that of control group (102.7+ 34.88 and 90.95+ 20.90 pg/ml, respectively, vs 62.136+ 18.19, Pw0.0001). The association between IL-4 level and SA has been explained by Helmby et al., who demonstrated that IL-4 is produced by basophils that have been stimulated and/or activated by interleukin 3, which is produced in large amounts as a result of the anaemia caused by the disease.19,20 Moreover, our results revealed that elevation of IL-4 level is correlated with parasitaemia severity (Spearman’s rho correlation coefficient was {0.360, twotailed significance50.0001) regardless of complications, as uncomplicated malaria group recorded the highest IL-4 mean level (62.136+ 18.19). Although our findings differ from what had been reported by Cabantous et al.20 who revealed that IL-4 concentrations were significantly higher in children with SM (median, 32 pg/ml) than in children with UM (median, 15 pg/ml) (P50.003) and that reported in the study of Nmorsi et al.21 who observed increased concentration of IL-4 in moderate infection (11.7 pg/ml) than in mild infection (1.6 pg/ml) (x2510.0, Pv0.05), our findings may ensure the conclusion that the elevation of IL-4 is correlated with hyperparasitaemia regardless the severity of the disease. This is in concordance with the fact that IL-4 may reduce the immunity to blood-stage parasites and thereby contribute to the growth of parasite mass.22 Thus, our study supports the view that IL-4 may be a risk factor for severe malaria20 and that IL-4 determines the degree of malaria parasitaemia, level of anaemia and clinical severity.

Conclusion Our results suggest that elevation of serum IL-4 in Sudanese children suffering from P. falciparum malaria is correlated with the severity of malaria hyperparasitaemia rather than with the severity of the disease.

Disclaimer statements Contributors ABE: concept and design of the study, data collection, laboratory investigations, statistical

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analysis and literature search. MH: data collection, analysis and data acquisition. AN: data collection, laboratory investigations. OEF: data collection and data acquisition. WB: data collection, clinical studies and data acquisition. NER: concept and design of the study, co-supervision of the study, clinical studies, analysis and interpretation. SA: concept and design of the study, analysis and interpretation, manuscript preparation and literature search. IA: data collection and data acquisition. MHFS: concept and design of the study, supervision of the study, clinical studies, analysis and interpretation, manuscript preparation, critical and final revision of the manuscript and literature search. Funding None. Conflict of interest There are no conflicts of interest. Ethics approval All patients were assured that all their obtained information will be handled in a confidential atmosphere and it will not affect their life after taking verbal and written consent. All the human studies were carried out according to the guidelines of the Animal and Human Ethical Committee of Omdurman Islamic University.

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18 Eisenhut M. The role of interleukin-4 in the immune response to Plasmodium falciparum. Parasite Immunol. 2010;32:470–1. 19 Helmby H, Kullberg M, Troye-Blomberg M. Expansion of IL-3-responsive IL-4-producing non-B non-T cells correlates with anemia and IL-3 production in mice infected with blood-stage Plasmodium chabaudi malaria. Eur J Immunol. 1998;28:2559–70. 20 Cabantous S, Poudiougou B, Oumar AA, Traore A, Barry A, Vitte J. Genetic evidence for the aggravation of Plasmodium falciparum malaria by interleukin 4. J Infect. Dis. 2009;200:1530–9. 21 Nmorsi OPG, Isaac C, Ukwandu NCD, Ohaneme BA, Eifediyi RA, Obiazi HAK. Serum anti-inflammatory interleukin profiles in Nigerian pregnant women infected with Plasmodium falciparum malaria. Arch Appl Sci Res. 2010;2:296–301. 22 Kumaratilake LM, Ferrante A. IL-4 inhibits macrophagemediated killing of Plasmodium falciparum in vitro: a possible parasite-immune evasion mechanism. J Immunol. 1992;149:194–9.

Correlation of interleukin-4 levels with Plasmodium falciparum malaria parasitaemia in Sudanese children.

This study aimed to measure the level of interleukin-4 (IL-4) in the serum of children patients with falciparum malaria and to correlate the productio...
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