International Journal of Academic Health and Medical Research (IJAHMR) ISSN: Vol. 3 Issue 1, January – 2019, Pages: 15-19 www.ijeais.org/ijahmr 15 Association Between IL-10 Levels With Plasmodim Falciparum Related to Age Groups and Density of Infection Among Sudanese PatientsKhartoum StateSudan 1 Ibrahim Mohammed Eisa, 2 Tayseer Elamin Mohamed Elfaki, 3 Mohamed Mobarak Elbasheir, 4 Mohammed Ahmed Ibrahim 1Department of Parasitology and Medical Entomology, Faculty of Medical Laboratory Science ALzaiem ALazahari University, Khartoum North, Sudan ibrahim.eisa1973@gmail.com 2Department of Parasitology and Medical Entomology, College of Medical Laboratory Science Sudan University of Science and Technology, Khartoum, Sudan tayseeralfaki5@gmail.com 3Department of Parasitology and Medical Entomology, Faculty of Medical Laboratory Science ALzaiem ALazahari University, Khartoum North, Sudan 4Department of Microbiology and immunology, Faculty of Medical Laboratory Science ALzaiem ALazahari University, Khartoum North, Sudan Abstract: A complex parasite such as human Plasmodium is likely to generate a variety of substances that injure the hosts directly or cause immunopathology. In malaria, a blood concentration of anti-inflammatory cytokines, such as interleukin (IL-10) is increased. The present study was performed to analyze IL-10 levels in patients with malaria falciparum and healthy controls individuals and correlate with malaria density infection as well as age groups. It is a cross sectional study was carried out in Khartoum state /Sudan, a total of 70 febrile patients and 20 healthy volunteers as controls were included in this study. Blood samples were collected from both patients and control candidates in (EDTA) containers for parasitological and immunological tests. Immunochromatography test and microscopic examination for both thick and thin blood films were used to detect the positive samples for Plasmodium falciparum. Enzyme linked Immunosorbant Assay (ELISA), was used for the determination of IL-10 levels. The result showed that of IL-10 levels in malaria patients were 1598.50 and SD 4771.64 while the levels in healthy participants the level was 10.39 and SD was 124.33 the difference was found to be statistically significant (P= 0.007). The levels of cytokines mentioned above were observed to be raised in malaria individual compared to healthy control candidates. Keywords-IL-10; P.falciparum; ELISA; Parasite Density; Khartoum State 1. INTRODUCTION Malaria remains a major global health threat in the 21st century. The number of human infections continues to increase in countries where the disease is endemic as well as in regions where the disease is not endemic [1]. The distribution and severity of malaria depends on the interaction of a number of factors. These include the size of the infective dose of sporozoites, nutritional status of the host, level of acquired immunity, host genetic factors, parasite growth rate, drug resistance status, socioeconomic conditions, standard of health care and education [2]. In areas with lower transmission, infections are less frequent and a larger proportion of the older children and adults have no protective immunity. In such areas, malaria can be found in all age groups. However, in areas with higher transmission, newborns will be protected during the first few months of life by maternal antibodies transferred through the placenta. As these antibodies decrease with time, young children (usually< 5 years of age become vulnerable to disease and consequently death by malaria [3]. Cytokines released during malaria may play both protective and pathological roles. Cytokine production is triggered via schizont rupture leading to release of substances/molecules such as parasite antigens, merozoites, pigment, glycosylphosphotidyl inositol anchor (GPI), and other soluble antigens or toxins that may induce cytokine production. Various cytokines have been shown to take part in malaria protection and/or pathogenesis. These include both the pro-inflammatory cytokines; IFN-γ, IL-1, IL-6, TNF-or anti-inflammatory cytokines, including IL-4, IL-10 and IL-5. The balance between proand anti-inflammatory cytokines may determine disease severity. IL-10 overproduction in response to TNF-α may play an important role in severe malarial anemia, but not apparently through down-regulation of TNF-α production [4,5]. Overall, an appropriate production of IL10 is required to protect against the harmful effects of TNF-α, thereby reducing the possibility of severe anemia and mortality induced by the malaria. Potential protective effects of IL-10 were observed in adult Kenyan volunteers whose peripheral blood mononuclear cells, in response to the liver stage antigen 1 (LSA-1), produced increased levels of IL-10, which International Journal of Academic Health and Medical Research (IJAHMR) ISSN: Vol. 3 Issue 1, January – 2019, Pages: 15-19 www.ijeais.org/ijahmr 16 correlated with subsequent resistance to re-infection with P. falciparum [6]. 2. Materials and methods 2.1 Study design: This is a cross sectional study. 2.2 Study area and study period: The study was conducted in Khartoum state-Sudan during period from July 2011 to July 2014. 2.3 Study population: A total of 70 febrile patients (cases) and 20 ages, sex matched healthy controls were included in this study. 2.4 Inclusion criteria: Patients with history of fever proceeding in the past 24 hours with confirmed malaria parasite positive slides and ICT were included study participant's cases and healthy volunteers were included as the study controls. Patients without recent (at least 2 weeks prior to examination). 2.5 Exclusion criteria: All patients who were negatively diagnosed for malaria parasites. Patients with any known concurrent chronic illness such as malnutrsion, concurrent infection, tuberculosis or pneumonia. Patients who were on antimalarial drug treatment during the last two weeks. 2.6 Sample size: The classical statistical method for determining sample size based on an unknown proportion of markers was used at 95 percent confidence level and 10 percent precision. Since there is no known figure for the prevalence of malaria, a prevalence rate of 50 percent was used to calculate the sample size using the formula below as described by Mendenhall et al. (1981) [7] . Or N = Z2 P (100 – P)/ d2 Where n = sample size P = prevalence rate Z = 1.96 at α = 0.05 (α = desired confidence level) d = desired width of confidence (precision) Q = 100-P There for the sample size (n) was determined as: n = 96.04 69 3. Methods 3.1 Sample collection and processing: Capillary blood for thick and thin film and intravenous blood for serological tests was collected from each candidate. 3.2 Collection of samples: Blood was collected aseptically after the thumb was cleaned with 70% alcohol before pricking. For thick and thin film, blood was collected from the tip of the finger by pricking with a needle. After that blood was allowed to ooze out one drop of blood was collected on a clean, dust and grease free glass slide. For serological tests, 2.5ml venous blood was collected into the collection tube containing anticoagulant (EDTA) by vein puncture and then processed [8,9]. 3.3 Preparation of thick film: After collection of blood on a clean and grease free glass slide, thick film was made by spreading one drop of blood with a spreader evenly on an area about 15×15 mm in diameter. Care was taken to avoid rouleaux formation. Then, the slide was labeled properly and allowed to air-dry by keeping the slide on horizontal position. Precaution was taken during spreading and drying [8]. 3.4 Preparation of thin film: After collection of one drop of blood on a clean grease free slide, thin film was made by spreading the blood using a smooth edged slide or spreader at an angle of 45o from the horizontal plane. A well-prepared thin blood film was judged by having a smooth tail end and free of vertical lines and holes. The slide was then labeled properly and allowed to air-dry [8]. Absolute methanol or ethanol was used to fix the thin film. Following steps were taken for fixing the thin film as described by Cheeshbrough (1999) [8]: The slide was placed horizontally on a staining rack. A small drop of absolute methanol or ethanol was applied to the thin film. Then the slide was allowed to fix for 1-2 minutes. Staining of the films: The slide was first placed on a staining rack. Then 10% Giemsa stain having a pH of 7.2 was poured gently on the fixed thin film or de-hemoglobinized thick film until the slide was totally covered. Then the slide was allowed to stain for 30-45 minutes out of the sunlight. Then the stain was washed with clean water. Back of the slide was wiped and placed in a draining rack. The slide was then allowed for airdry [8]. 3.5 Microscopic examination of the stained film Thick film: The thick film was examined first by using the 40x objective, a well-stained part of the thick film was selected which was well populated with WBC. Then the selected portion of the film was examined with 100x oil immersion objective by moving along the width of the slide. At least 100 fields were examined before a slide was considered as negative for malaria parasite [8]. Thin film: The thin film was examined by the 40x objective first followed by 100 x oil immersion objectives. After applying immersion oil the film was examined by moving along the edge of the film. Then moving the slide inward by one field, returning in a lateral movement and so on. At least 100 fields were examined before a slide was considered as negative for malaria parasite [8]. 3.6 Determination of malaria parasitaemia: International Journal of Academic Health and Medical Research (IJAHMR) ISSN: Vol. 3 Issue 1, January – 2019, Pages: 15-19 www.ijeais.org/ijahmr 17 Parasites density determination: The method used is based on the number of parasites per μ1 of blood in a thick film which are counted in relation to a predetermined number of leucocytes. An average of 8000 leucocytes per μ1 is normally taken as the standard. Two tally counters were used to count parasites and leucocytes separately. In each case, the parasites counts in relation to leucocyte count were expressed as parasites per μ1 by the following formula as described by WHO (2010) [10]. 3.7 Detection of Antibody: Antibody was detected by immunochromatographic (ICT) method with Malaria P. falciparum .P.vivax P.ovale and P.malariae Antibody onsite Rapid Screening Kit. The kit was manufactured by Standard Diagnostics, Inc. Korea. Lot no: 044013. Test procedure performed according to manufactories instructions. Principle of the test: The Malaria onsite Rapid test is a one step chromatograhic immunoassay which specifically detects the antibodies to P. falciparum, P.vivax, P.ovale and P. malariae in human serum or plasma. The test utilizes P .falciparum specific antigen HRP-II and P.vivax antigen aldolase to ensure test specificity and sensitivity. Sample collection for serology: Two and half milliliters (2.5 mls) of venous blood were collected in EDTA containers at the time of recruitment of participants for determination of cytokines. The blood was then centrifuged immediately and plasma was collected and stored at – 20o C until analysis. 3.8 Determination of IL-10: IL-10 levels expression were determined using specific monoclonal antibodies to IL-10 using sandwich ELISA at 450 nm wavelengths, according to the manufacturer's instructions (RF). 3.9 Ethical consideration and permission: Ethical approval was sought from the University of Alzaiem Alazhari and individual patients and the purpose and benefit of the study were explained. 3.10 Data analysis: Data was first entered in Microsoft excel, then converted and analyzed using SPSS 15.0. 4. Results The results showed that IL-10 levels in malaria patients was 1598.5 and SD was 4771, while the levels in healthy participants was 10.39 and SD was 124.33.The difference was found to be statistically significant (P= 0.007) (Table 1). The results showed that there was positive relation between the parasite density and IL-10 as the correlation coefficient (rho) was found to be +0.072 and the result was found to be statistically insignificant at P=0.552 (Table 2) implied that when the parasite density was high the IL-10 was high (Figure 1). The results showed that there was positive relation between IL-10 and age groups as the correlation coefficient (rho) was found to be + 0.144 and the result was found to be statistically insignificant at P=0.235 (Table 3) showing that the levels of IL-10 was increasing by age increased (Figure 2). Table 1: Mean and SD levels of IL10 in patients and controls individuals Group Sample IL-10 (pg/ml) P.value Patients Mean SD 1598.50 4771.64 0.007 Control Mean SD 10.39 4771.64 Table 2: Correlation between parasite density and IL-10 Parasite Density (parasites/μl) P.value IL-10 + 0.072 (rho) 0.552 Figure 1: Correlation between parasite density and IL-10 Table 3: Correlation between IL-10 and age groups Age groups P.value L-10 +0.144 (rho) 0.356 Figure 2: Correlation between IL-10 and age groups Series1, No malaria, 0 20000 2000 1500 1772.03 1819.75 176.13 International Journal of Academic Health and Medical Research (IJAHMR) ISSN: Vol. 3 Issue 1, January – 2019, Pages: 15-19 www.ijeais.org/ijahmr 18 5. Discussion The study aimed to determine the existence of a correlation between IL-10 cytokine and malaria infection. The role of cytokines in the pathogenesis of malaria has been clearly confirmed, but not completely understood. It is thought that many factors impact on their level, which in the course of malaria depends on the circadian rhythm, and in accordance with this, there are large variations in the cytokine levels. The anti-inflammatory cytokines, IL-10 levels were elevated in patients with malaria compared to healthy controls; older patients had much greater elevated levels of IL-10 compared to the other age groups. The higher levels of IL-10 in older patients could be due to their ability to mount strong T-helper type 2 responses quite early in infection. Experimental malaria studies performed exclusively in adult hosts indicated that both CD4+ and CD8+ T cells play an important role in the defense against malaria. To address the role of these cells in an age-dependent model, where young susceptible rats can be protected by the transfer of whole spleen cells from adult protected rats, TCR cells were transferred to young infected rats. These experiments indicated that 58% of young rats recovered from infection after T cell transfer [11]. Furthermore, IL-10 has an important role in imunoregulation down regulation of cytokines production (predominantly TNF-α, IL-6 and IL-12), which have Th1 function and as a result, Th1 functions are inhibited and natural killer cell activity is promoted [12]. This finding is consistent with our suggestion that the balance between the Th1 cytokine TNF-α and the Th2 cytokine IL-10 is critical in the pathogenesis of severe disease in P. falciparum-infected persons who live in areas in which malaria is endemic. Consistent with the present results, study carried by Linke et al. (1996) [13] showed that IL-10 gene knockout mice with an intrinsic deficiency for IL-10 production when infected with Plasmodium chabaudi chabaudi succumb to severe disease and higher mortality than their heterozygote counterparts or normal mice. An enhanced Th1 response persisted throughout the course of infection in the IL-10deficient mice, while in control mice a Th2 response was predominant. In another study carried by Kossodo et al. (1997) [14], the exogenous administration of IL-10 to susceptible CBA mice protected them from Plasmodium berghei-induced cerebral malaria while an in vivo neutralization of IL-10 in resistant BALB/c mice induced a neurologic syndrome. Collectively, these findings support the hypothesis that IL-10 is a critical factor in downregulating the pathogenesis of severe malaria. The younger children appear not to maintain IL-10 levels in response to inflammatory process, hence, probably the observed relative lower levels of cytokine compared to their older counterparts. This in turn may explain the younger children's relative higher susceptibility to anemia as reported by Nussenblatt et al. (2001) [15] as they do not mount enough Th2 response to counteract the suppressive erythropoiesis effects of TNF-α. 6. Conclusion: This study concluded that there was positive relation between IL-10 level and malaria infection among Sudanese patients related to age groups, showing that the levels of IL-10 was increasing by age increased. References [1]Palmer, C. J., Bonilla, A., Bruckner, D. A., Barett, E. D., Miller, N. S. & Haseeb, M. A. (2003). Multicenter study to evaluate the optimal test for rapid diagnosis of malaria in U.S. Hospitals. Journal of Clinical Microbiology, 41 (11), 5178-5182. [2]Phillips, R. S. (2001). Current Status of Malaria and Potential for Control. Clinical Microbiology Reviews, 14 (1), 208-226. [3]Center for Disease control and Prevention (CDC) (2004). The History of Malaria an Ancient Disease. [4]Kurtzhals, J. A., Adabayeri, V., Goka, B. Q., Akanmori, B. D., Oliver-Commey, J. O., Nkrumah, F. K., Behr, C. & Hviid, L. (1998). Low plasma concentrations of interleukin 10 in severe malarial anaemia compared with cerebral and uncomplicated malaria. Lancet, 351, 1768-72. [5]Othoro, C., Lal, A. A., Nahlen, B., Koech, D., Orago, A. S. & Udhayakumar, V. (1999). A low interleukin-10 tumor necrosis factor-alpha ratio is associated with malaria anemia in children residing in a holo-endemic malaria region in western Kenya. Journal of Infectious Diseases, 179, 279-82. [6]Kurtis, J. D., Lanar, D. E., Opollo, M. & Duffy, P. E. (1999). Interleukin-10 responses to liver-stage antigen 1 predict human resistance to Plasmodium falciparum. Infectious Immunology, 67, 3424-9. [7]Mendenhall W., R. L. Scheaffer, and Wackerly, D.D. (1998). Mathematical Statistics with Applications. Duxbury Press, Boston. Pp 375. [8]Cheesbrough, M. (1999). District Laboratory practice. In: Tropical Countries, Part-1, Low price edition. Bath press and bounded in at the University press, Cambridge, 239-258. [9]White, N. J. (2003). Malaria. In: Gordon C, Cook and Zumla A, editors. Mansons Tropical Diseases, 21st edition. Saunders Elsevier Science, Health Science Division, London, 1205-1295. [10]World Health Organization (WHO) (2010). Basic malaria microscope2nd edition, ISBN 978 92 4 154782 6 (Part 1). [11]Paterson, D. J., Jefferies, W. A., Green, J. R., Brandon, M. R., Corthesy, P., Puklavec, M. & Williams, A. F. (1987). Antigens of activated rat T lymphocytes including a molecule of 50,000 Mr detected only on CD4 positive T blasts. Mollecular Immunology, 24, 1281-1290. [12]Lyke, K. E., Burges, R., Cissoko, Y., Sangare, L., Dao, M. & Diarra, I. (2004). Serum levels of the proInternational Journal of Academic Health and Medical Research (IJAHMR) ISSN: Vol. 3 Issue 1, January – 2019, Pages: 15-19 www.ijeais.org/ijahmr 19 inflammatory cytokines interleukin-1 beta (IL-1beta), IL-6, IL-8, IL-10, tumor necrosis factor alpha and IL-12 (p70) in Malian children with severe Plasmodium falciparum malaria and matched uncomplicated malaria or healthy controls. Infectious Immunology, 72, 5630-5637. [13]-Linke, A., Kuhn, R., Muller, W., Honarvar, N., Li, C. & Langhorne, J. (1996). Plasmodium chabaudi chabaudi: differential susceptibility of gene-targeted mice deficient in IL-10 to an erythrocytic-stage infection. Experimental Parasitology, 84, 253-63. [14]Kossodo, S., Monso, C., Juillard, P., Velu, T., Goldman, M. & Grau, G. E. (1997). Interleukin10 modulates susceptibility in experimental cerebral malaria. Immunology, 91, 536-40. [15]Nessenblastt, V., Makasa, G., Metzger, A., Ndeezi, G., Garratt, E. & Semba, R. D. (2001). Anemia and Interleukin10, TNF-α and erythropoietin levels among children with acute, uncomplicated Plasmodium faliciparum malaria, Clinical and Diagnosis Laboratory. Immuonology, 8, 11641170 .