Maternal Expressions (Serum Levels) of Alpha Tumour Necrosis Factor, Interleukin 10, Interleukin 6 and Interleukin 4 in Malaria Infected Pregnant Women Based on Parity in a Tertiary Hospital in Southeast, Nigeria

1 Department of Medical Laboratory Science, Imo State University, Owerri, Imo State, Nigeria. Department of Nursing Science, Ebonyi State University, Abakaliki, Ebonyi State, Nigeria. 3 Department of Physiotherapy, Evangel University, Akaeze, Ebonyi State, Nigeria. 4 Department of Haematology and Immunology, Faculty of Clinical Medicine, Ebonyi State University, Abakaliki, Nigeria. 5 Department of Public Health Education, Gregory University, Uturu, Abia State, Nigeria. Department of Medical Laboratory Science, Ebonyi State University, Abakaliki, Ebonyi State, Nigeria.


INTRODUCTION
Malaria has been reported as a condition caused by infestation with Plasmodium parasite species, is a significant public health problem globally especially in developing countries like Nigeria causing considerable morbidity and mortality especially in sub-Saharan Africa where it accounts for up to 1 million death annually [1]; [2]. Dellicour et al. [3] reported that pregnant women are susceptible to malaria infection. Malaria during pregnancy is a major public health challenge in endemic tropical countries, like in sub-Saharan Africa. Desai et al. [4] opined that about 125 million pregnant women live in malaria endemic areas in sub-Saharan Africa, and 32 million of these pregnant women are at threat of malaria.
Pregnant women are at high threat of being infected with malaria owning to the capacity of the parasite to stick to trophoblastic villous epithelium and sequester in the placenta, which could eventually lead to poor pregnancy outcome [5]. It revealed that over 200,000babies die yearly in sub-Saharan Africa because of their mother becoming infected with malaria during pregnancy [6]. Malaria during pregnancy can lead to maternal and foetal adverse effects, mainly anaemia, cerebral malaria, hemorrhage, and low birth weight.
Cytokines are low molecular weight regulatory proteins that are secreted by many cells of the immune system in response to several stimuli [7].
They are involved in virtually all physiological responses in the body and are critical players in coordinating cell-to-cell communication between immune cells; by binding to specific cell membrane receptors inducing induce cellspecific immune responses. They are secreted by many cells of the immune system in response to a number of stimuli. During successful pregnancies, fetal trophoblasts and maternal leukocytes secrete predominantly T-helper 2 type cytokines to prevent initiation of inflammatory and cytotoxic type responses that might damage the integrity of the materno-fetal placental barrier [8]. In response to invading malaria parasites, however, Th-1 type cytokines are produced to reverse the Th-2 type bias within the placenta [9]. Inconsistence reports on the response of some pro-inflammatory cytokines to peripheral and placental malaria have been reported [10]; Diouf et al. [11]. Both pro and anti inflammatory cytokines are found at significantly increased levels in the peripheral blood and the intervillous spaces of the placentas of malaria-infected women. The Production of these cytokines is responsible for the resulting Th-1: Th-2 imbalance observed in Plasmodium falciparuminfected placentas [12,5].
Severe malaria has long been associated with high circulating levels of inflammatory cytokines such as tumour necrosis factor (TNF-a), IL-1, IL-6. Studies have demonstrated a link between TNF-a, IL-6, IL-10 and the severity of the disease in human malaria [13]. Anti-inflammatory cytokines have also been found to have important roles in the immune response against Plasmodium. IL-10 exerts a vital role as an immunoregulator during Plasmodium falciparum infection, neutralizing the effect of the other cytokines produced by Th-1 and CD8 cells [14], [15]. Additionally, IL-10 and granulocyte colonystimulating factor (G-CSF) is elevated and correlated with parasitaemia in asymptomatic pregnant women in Ghana [16], suggesting that these cytokines may act to reduce symptoms.
A study was done to determine the maternal serum levels of alpha tumour necrosis factor, interleukin 10, interleukin 6, and interleukin 4 in malaria-infected pregnant women based on their parities in Southeast, Nigeria.

Study Area
This study was carried out in Federal Medical Centre Umuahia in Abia State, Nigeria.

Subjects
A total of 150 subjects between the ages of 18-45 years were recruited for the study comprising fifty (50) subjects for each of the 3 parities (Groups, A-C).

Experimental Design
A prospective cross-sectional study was carried out in 3 groups.
Group A =50 Malaria Infected Pregnant Subjects at prime parity, Group B =50 Malaria Infected Pregnant Subjects at second parity, Group C =50 Malaria Infected Pregnant Subjects a multi-parity (three or more parities).
Oral consent was obtained from the patients after which a structured questionnaire was administered to all respondents who were also part of a clinical study, and the subjects were allowed to join in the study voluntarily and can withdraw at any stage of the study.

Exclusion Criteria
Those excluded from the study were:  Pregnant women with evidence of chronic infection like HIV, tuberculosis and inflammatory disease;  Women who did not give their informed consent;  Pregnant women needing emergency care or having an at-risk pregnancy such as gestational diabetes, pre-eclampsia and eclampsia.

Sample Collection
Eight milliliters (8 ml) of venous blood were drawn from each participant using standard veno puncture techniques.
2.5 ml was dispensed in EDTA container for malaria detection, and 5.5 ml were dispensed into a plain container to obtain serum. The sample in the plain test tube was allowed to clot at room temperature and centrifuged to separate the serum.

Laboratory Procedures
All reagents were commercially purchased, and the manufacturer's Standard Operating Procedures (SOP) were strictly followed.

Malaria estimation using rapid test kit [17]
As modified by SD BIO LINE One Step Malaria antigen P.F (HRP-II) rapid kit was used.
Test procedure: The kit was allowed to equilibrate at room temperature. The test device was opened for and labeled for each patient. The specimen was collected with the aid of a capillary pipette provided and then transferred into the round specimen well. Four drops of assay diluents were dispensed into the diluents well. The kit was left on a flat bench for a period of 15 minutes before taking the result.

Malaria parasite identification using Giemsa Staining Technique [18]
Methodology: A drop of blood was placed on the slide to cover the diameter of15-20 mm. The blood was smeared evenly on the slide to obtain a thick film and then allowed to air dry with the slide in a horizontal position. Before staining, the stock Giemsa stain was diluted in 1:10 dilution using phosphate buffer at pH 7.2. The working solution of the Giemsa stain was used to cover the dried thick film for 30 minutes, and at the end of the staining period, water was used to flush the stain off the slide. The slide was rinsed briefly in gently running tap water, and the undersurface of the slide blotted dry to remove excess stain. It was left to air dry in a vertical position and then viewed microscopically using x40 and x100 objectives.

Alpha tumour necrosis factor (TNF-α) assay
Human Alpha Tumour Necrosis Factor Commercial ELISA Kit by MELSIN Medical Co Limited was used. Catalogue Number: EKHU-0110 Procedure: Dilutions of standard were prepared to get a concentration of 80 pg/mL, 40 png/mL, 20 pg/mL, 10 pg/mL, 5 pg/mL and 0 pg/mL. 50 uL of standards were pipette into the standard wells. 10 uL of test serum were pipette into each sample well. 40 uL of sample dilluent was added to the sample well. Sample blank was included (to contain only chromogen solution A and B, and stop solution). 50 uL of HRP-conjugate reagent was added to all wells except blank, covered with an adhesive strip and incubated for 30 minutes at 37 o C. It was washed four times. 50 uL of chromogen solution A and 50 uL of chromogen solution B was added to each well. They were mixed incubated for 10 minutes at 37 o C. 50 uL of stop solution was added to each well. Optical densities of the samples were read in a microtiter plate reader at 450 nm wavelength within 15 minutes taking the blank well as zero concentration.

Statistical Analysis
All statistical analysis was performed using SPSS version 20. The results were expressed as mean plus or minus standard deviation in tabular form. The student t-test was used for comparison of differences in various groups. All tests performed were two-tailed and the level of significance significant was set at p<0.05.

DISCUSSION
The study showed no changes in the cytokines studied among the malaria infected pregnant women based on their parities. Cytokines are immune regulators that are vital to the response in different conditions. Pregnancy is known to exert stress on women and changes in immunological systems. The cytokines studied are inflammatory and anti-inflammatory. A balance has to be established between these cytokines. This study shows that malaria infection does not change these cytokines in pregnant women based on their parities. Malaria infection has long been associated with high circulating levels of inflammatory cytokines such as TNF-a, IL-1, IL-6. Studies have demonstrated a link between TNF-a, IL-6, IL-10 and the severity of the disease in human malaria [13]. Anti-inflammatory cytokines have also been found to play essential roles in the immune response against Plasmodium. IL-10 has an important role as an immunoregulator during plasmodium falciparum infection, neutralizing the effect of the other cytokines produced by Th-1 and CD8 cells [14,15]. These cytokines are known to be elevated in malaria infected pregnant women as reported in the study of Okorie et al. [14]. Interleukin 10 (IL-10) and IL-4 modulate TNF alpha, act exerting immunoregulatory role during pregnancy by impeding the inflammatory response. Cytokines have a significant role in the pathogenesis of malaria and their levels can be useful as diagnostic markers for malaria and for monitoring the severity of the disease [14]. In a study carried out by Obeagu and his research team, there were no changes in these cytokines studied among the malaria infected pregnant women based on gestational ages except when IL-10 was compared between the subjects on second trimester and third trimester [7].

CONCLUSION
The study showed no changes in the cytokines studied among the malaria infected pregnant women based on parities. This study shows that malaria infection does not change these cytokines in pregnant women based on their parities.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but the advancement of knowledge. Also, the research was not funded by the producing company, instead rather it was funded by the personal efforts of the authors.

CONSENT
Informed consents obtained from the participants were recruited among pregnant women booked for antenatal care in the hospital.

ETHICAL APPROVAL
As per international standard or university standard written ethical approval has been collected and preserved by the author(s).