Asymptomatic malaria infection among children with Sickle Cell Anaemia: The role of IL-10 and possible predisposing factors

Asymptomatic malaria infection among children with Sickle Cell Anaemia: The role of IL-10 and possible predisposing factors

Idayat Ajoke Oyetunji^1,&, Abimbola Joseph Amoo², Titilope Adenike Adeyemo³, Oyesola Oyewole Ojewunmi¹

¹Sickle Cell Foundation Nigeria, National Sickle Cell Centre, Opposite Lagos University Teaching Hospital, Lagos, Nigeria, ²Department of Medical Microbiology and Parasitology, Obafemi Awolowo College of Health sciences, Olabisi Onabanjo University, Ago-Iwoye, Nigeria, ³Department of Haematology and Blood Transfusion, Faculty of Clinical Science, College of Medicine, University of Lagos, Lagos University Teaching Hospital, Idi-Araba, Lagos, Nigeria

^&Corresponding author
Idayat Ajoke Oyetunji, Sickle Cell Foundation Nigeria, National Sickle Cell Centre, Opposite Lagos University Teaching Hospital, Lagos, Nigeria

Introduction    

Malaria is one of the prevailing human parasitic diseases, ranking first in terms of its socio-economic and community health burden in tropical and subtropical areas, in sub-Saharan African and South-west Asian countries [1-3]. Plasmodium falciparum and Plasmodium vivax signify the majority of infections amongst the five Plasmodium species that are capable of causing human infection; the former is accountable for the greater part of malaria linked mortality [4]; vivax malaria which was regarded as a benign disease, has currently and obviously emerged as a potentially fatal state [5], predominantly in non-African endemic regions. Plasmodium infection has a broad range of signs that are grouped into three major clinical groups: asymptomatic (presence of malaria parasite devoid of symptoms), mild and severe [6, 7]. Individuals with asymptomatic Plasmodium infection can remain infected for lengthy episodes [8]; they eventually build up symptomatic disease if they have a dysregulated response to the immune system. Asymptomatic cases offer a basic reservoir of parasites and they may become gametocyte carriers contributing to the persistent spread of malaria; parasites from the asymptomatic carrier could be more infectious than the symptomatic ones [7,9]. In Human malaria, the relationship between TNF-α, IL-6, IL-10 and disease severity has been documented [10]. At diagnosis and even in cases of uncomplicated malaria infection, raised plasma levels of these cytokines have been established [11,12]. IL-10, an essential regulator of immune response during Plasmodium falciparum infection plays its role by reducing the effects of other cytokines formed by T-helper (TH) 1 and CD8 cells [13,14]. IL-10 has been found to be high and is linked with parasitaemia in individuals with asymptomatic malaria infection thus, signifying that this cytokine may possibly operate to decrease symptoms [6,11,15].

The diagnosis of asymptomatic malaria infection is not clear due to apparent lack of clinical manifestation and low level of parasites [16]. Although the gold standard for malaria diagnosis is microscopy, its detection limits, subjective nature of the diagnosis which depends on the training and experience of the microscopist, permit a variance in accurate diagnosis. However, malaria rapid diagnostic test (RDT) which is based on parasite antigen detection using antibodies provides alternative to microscopy; this method also is not without its limitations in terms of its sensitivity and specificity in malaria infection detection. Polymerase Chain reaction (PCR)-based diagnostic method of malaria infection has been proven reliable and more accurate in malaria diagnosis. PCR-based method detected the prevalence of 19.2% of sub-microscopic P. falciparum carriage in RDT and microscopy negative samples indicating the sensitivity and specificity of PCR in malaria detection [17]. In malaria endemic areas, asymptomatic malaria infection is widespread and has become a serious concern as efforts are rising towards eliminating the parasite [18]. Despite the implication of socioeconomic conditions in the distribution of asymptomatic malaria infection as well as the susceptibility of children, few studies have explicitly focused on the need to further evaluate the burden of asymptomatic malaria infection using more sensitive method (PCR) as well as the role of IL-10 among children living with sickle cell disease (SCD) to scale up elimination and eradication in Nigeria. Globally, about 85% of all children with SCD occur in Africa in which about 150,000 children are born in Nigeria every year [19]. To this end, malaria is frequently observed as a major problem for African children living with SCD; hence, they are given appropriate malaria prophylaxis [20]. Plasmodium falciparum has also been reported as a characteristic or distinctive factor of painful crisis in people living with SCD residing in endemic countries [21,22]. Therefore, this study will explore the role of IL-10 levels and possible risk factors in asymptomatic malaria infection and make a definite diagnosis of Plasmodium infections.

Methods     

Recruitment: this study comprised sixty children with SCD (in steady state) and forty children (the siblings living without SCD as well as other apparently healthy individuals from the community without SCD) as the control group, aged 2-16 years. The study was carried out at the Sickle Cell Foundation Nigeria between March and May, 2016. Fifty-three (83.3%) of sixty subjects with SCD were on daily use of proguanil as malaria prophylaxis while none of the control subjects was on routine use of any malaria prophylaxis and they had not been treated for malaria three months preceding recruitment for this study. Steady state Hb SS refers to subjects with no history of illness such as infection, inflammation during the previous 4 weeks, no history of hospital admission 2-3 days after the point in time in question, no treatment with medications such as antibiotics that may affect the blood counts during the previous 3 weeks, those without history of an acute painful episode that required hospitalisation for at least four successive weeks after a previous painful crisis [8]. Subjects with or without SCD who had blood transfusion three months prior to the study recruitment were excluded. Any child with no recent history of symptoms and/or signs of malaria, axillary temperature of < 37^oC and shows laboratory confirmation of malaria infection was regarded as being asymptomatic [6,23].

Ethical consideration: ethical approval was obtained from the Ethics and Research Committee of Lagos University Teaching Hospital (HREC registration no: NHREC: 19/12/2008a). Additionally, a written informed consent was obtained from the parents/guardians and verbal assent from children older than 7 years of the study participants before sample collection.

Data and sample collection: a structured questionnaire based on possible predisposing factors for asymptomatic malaria was developed. Five millilitres of venous blood sample was collected from each participant, 3ml was dispensed into ethylenediamine tetra acetic (EDTA) tubes and the remaining 2ml was poured into a plain tube with clot activator. The blood sample in each EDTA tube was used for the RDT for Plasmodium falciparum and preparation of thin and thick blood films immediately after collection. The remaining was then stored at -20^oC for the molecular diagnosis of asymptomatic parasitaemia for six weeks. The blood sample in each plain tube was spun at 3000rpm for 10minutes immediately after collection. The serum was dispensed into cryovials and stored at -20^oC for six weeks for the measurement of IL-10 levels by ELISA technique.

Detection of malaria infection: this was achieved using RDT for Plasmodium falciparum detection and microscopic techniques using 10 % Geimsa stained fixed thin and unfixed thick blood films [24].

Quality control of microscopy: the films were made in duplicates, stained with good quality Geimsa stain following Standard Operating Procedures. One of the duplicate was read by an experienced medical laboratory scientist and the other read by a WHO certified Microscopist.

Molecular diagnosis of malaria infection: DNA preparation from blood: This was prepared using DNA extraction kit from Jena Bioscience (Spin column based genomic DNA purification from whole blood) following the Manufacturer´s instructions. DNA amplification by nested PCR: The PCR assays were performed using Applied Biosystem thermal cycler, 9800. The first nested PCR was performed in a 15 μl total reaction volume containing 1 μl DNA template, genus-specific primers (rPLU1 and rPLU5), 5X Taq master mix and PCR grade water. The PCR conditions were: initial denaturation at 92^oC for 2 min, 30 cycles of denaturation at 92^oC for 30 sec, annealing at 55^oC for 1 min and extension at 72^oC for 1 min. The amplification process was completed with a final extension at 72^oC for 2 min. Nested-2 PCR was performed in a 25 μl reaction volume using 1 μl of the nested-1 PCR product as a template DNA using the genus-specific (rPLU 3 and rPLU 4) and species-specific primer pairs (rFAL 1 + rFAL 2, rMAL 1 + rMAL 2, rOVA 1 + rOVA 2, rVIV 1 + rVIV 2). The cycling conditions were similar to the nested-1 PCR except that the annealing temperature was 62^oC for the genus-specific primers (rPLU 3 and rPLU 4) and 58^oC for the species-specific primers. The primers sequence and sizes of the PCR products have been previously described by Singh et al. (1999) [25]. The amplified products were run through 2% agarose gel stained with ethidium bromide. A low molecular weight DNA ladder (from Jena Bioscience, Jena, Germany) was used. The gels were visualized in a gel documentation system (Alpha imager, Alpha Innotech, USA).

Data analysis: data were entered and analyzed using SPSS 15.0. Descriptive statistics, student´s t-test and Pearson correlation coefficient test were carried out. P-values less than 0.05 were taken as being statistically significant.

Results     

Socio-demographic and clinical characteristics of the study participants: the mean±(SD) age of subjects with SCD was 6.76±(3.24) years (range 2-16 ) while the control group was 7.77±(3.85) years with P = 0.161. There was no significant difference in the level of education and monthly income between the study participants with SCD and the control group (P>0.05). However, the nature of the living facility of those with SCD differs significantly from those of the control group (P<0.0001). The mean±(SD) of body temperature of the study participants living with SCD was significantly lower compared to those without SCD (P = 0.037) (Table 1).

Asymptomatic malaria infection: among patients with SCD, two samples (3.3%) were positive for RDT, three samples (5%) were positive for microscopy, and four samples (6.7%) were positive for nested PCR technique. None of the patients in the control group was positive for Plasmodium falciparum detection using the three diagnostic methods. The frequency of asymptomatic malaria infection, however, using nPCR was not significant compared to the control group (P = 0.96) (Table 2). The two samples that were positive for RDT were also positive for microscopy but only one sample was positive for all the three methods examined in this study. It should be noted that none of the study subjects was positive for Plasmodium species other than Plasmodium falciparum during the PCR malaria detection assay.

Socio-economic risk factors of malaria infection among SCD group: the socio-economic risk factors of subjects with SCD who had asymptomatic malaria infection was not significantly different from those that were negative (P>0.05) (Table 3).

IL-10 levels and asymptomatic malaria infection in patients with SCD: IL-10 levels in subjects with SCD with asymptomatic malaria infection was significantly higher than those that were negative for malaria infection (9.14±1.08 vs 4.80±2.62). IL-10 values in pg/ml was significantly associated with asymptomatic malaria parasitaemia (P<0.0001). However, the mean±SD values among the control group that were not infected was higher than that of the SCD group that were negative for asymptomatic malaria parasitaemia (Table 4).

Discussion     

Sickle cell disease (SCD) contributes significantly to under five mortality in sub-Saharan Africa and malaria is commonly regarded as a major contributor in these children [20, 26-28]. Global distribution of HbS reflects the selective advantage of sickle gene which offers protection against Plasmodium falciparum for sickle cell carriers (Hb AS) in malaria endemic countries. The protective effect of HbAS against malaria infection differs significantly from HbSS, although prevalence of malaria parasitaemia is lower in patients with Sickle cell anaemia (SCA) than in patients without SCA, severe anaemia and mortality are remarkable in patients with SCA hospitalised with malaria [29, 30]. Hence, malaria chemoprophylaxis is offered routinely to persons living with SCD to prevent malarial infection and sickle cell crises. Substantial evidence has revealed that prophylaxis improves haemoglobin concentrations and reduces severe anaemia, the number of clinical malaria attacks, and mortality [31]. Asymptomatic malaria parasitaemia is suggestive of low densities of Plasmodium parasites (asexual or gametocytes) below the limit of microscopic detection and the carriers constitute a reservoir for malaria transmission. Use of insecticide treated-bed nets (ITN) for the control and prevention of malaria is remarkable with the decline in malaria related morbidity and mortality. The statistical significance in the frequency of patients with SCA who use ITN compared to the control in this study could results from the knowledge about malaria prevention strategies gained during genetic counseling. This is in agreement with the finding of Lengeler (2004) which reported effectiveness of ITNs in malaria prevention program in reducing childhood mortality and morbidity [32]. In malaria endemic regions, diagnosis of asymptomatic malaria infection is quite challenging. PCR assay, a DNA-based molecular diagnostic technique, has been found to be more sensitive and specific than RDT and microscopy [33]. The sensitivity, specificity, and positive predictive values of nPCR in comparison with microscopy were 100, 97.2, and 73.3% respectively [34]. Meanwhile, Golassa et al. (2013) reported sensitivity of PCR relative to microscopy and RDT as 90.7% and 80%, respectively with the overall PCR-based prevalence of Plasmodium falciparum infection of 5.6 and 3.3 fold higher than that detected by microscopy and RDT respectively [17]. They further showed the prevalence of Plasmodium falciparum infection of 3.7%, 6.9% and 20.6% using Microscopy, RDT and PCR-based detection methods among the Ethiopians with sub-microscopic Plasmodium infection.

Microscopy-based prevalence of asymptomatic malaria parasitaemia in apparently healthy children was 6.8% and 7.4% in Ethiopia and Zambia respectively but 25.6% was reported among primary school children in Osun state, Nigeria. No asymptomatic malaria case was detected among our control subjects indicating that they did not harbour asexual parasites and gametocytes in keeping with zero prevalence of asymptomatic malaria parasitaemia found among apparently healthy children in India and Tanzania [33, 35]. Although majority of our control subjects were not using insecticide treated bed nets, use of screened windows and doors with mosquito nets and indoor spraying of insecticide largely practised by the subjects could be responsible for the zero prevalence observed in our study. Microscopic malaria infection detection which showed 5% was much lower compared to 24% reported a decade ago in Ibadan, Nigeria [20]. The widespread and effective practice of the malaria control and prevention strategies of the patients may account for the lower frequency reported in this study. It has been shown that adolescents and adults are more likely to develop asymptomatic malaria due to a certain level of age-dependent immunity which develops [17,36]. Therefore, younger age of patients with SCD in our study (mean±(SD):7.2±(3.5) years) compared to young adults (mean age: 22 years) could also account for the low prevalence we reported. Among the patients with SCA, PCR-based prevalence of asymptomatic Plasmodium falciparum infection was 6.7% demonstrating superior diagnostic performance of PCR assay compared to RDT and microscopy. This prevalence reported among patients with SCA in spite of the fact that they were on malaria chemoprophylaxis (Proguanil) suggests that malaria infection is worth considering in outpatient clinic and during hospitalisation. IL-10 is an anti-inflammatory cytokine; its high levels coupled with tumor necrosis factor-alpha and lower levels of IL-12 suppress anti-parasitic cellular immune responses leading to severe malaria [37]. Higher IL-10 levels observed in patients with SCA who were positive for PCR-based asymptomatic Plasmodium falciparum detection is similar to other studies that reported relationship between clinical protection to malaria and high plasma IL-10 levels [6, 38]. Hugosson et al. (2004) observed that in children with asymptomatic malaria, clinical protection to malaria is connected with an increased in self-reactive response, low levels of Plasmodium falciparum-specific IgG, high plasma levels of interferon-gamma and IL-10. He further demonstrated that high IL-10 levels were associated with less-effective Plasmodium falciparum parasites clearance. High IL-10 levels in our patients are suggestive that those patients are more prone to slower parasite clearance [39]. Thus, proposing a hypothesis that there is an association between asymptomatic malaria and IL-10 levels in children living with SCD.

Conclusion     

This study demonstrated accurate diagnosis of asymptomatic malaria infection using PCR-based technique. We established that asymptomatic malaria is present in steady state SCA children and is associated with elevated IL-10 levels.

Competing interests     

The authors declare no competing interests.

Authors' contributions     

Idayat Ajoke Oyetunji: conceptualization; Oyetunji, Adeyemo, Amoo and Ojewunmi: design; Oyetunji and Ojewunmi: acquisition of data; Oyetunji and Ojewunmi: data analysis and interpretation; Oyetunji: article draft; Oyetunji, Adeyemo, Amoo and Ojewunmi: article review. All the authors have read and agreed to the final manuscript.

Acknowledgments     

We thank all children and their parents/ guardians for their participation in this study. We would also like to say a big thank you to Mr Samuel, WHO certified microscopist from the Nigerian Institute of Medical Research, Lagos, Nigeria for reviewing the prepared slides.

Tables      

Table 1: socio-demographic and clinical characteristics of the study participants

Table 2: prevalence of Asymptomatic malaria infection among the study participants

Table 3: socio-economic risk factors of malaria infection among SCD group

Table 4: IL-10 level and asymptomatic malaria infection

References     

1. World Health Organization (WHO). World Malaria report. WHO Library Cataloguing-in-Publication Data. 2011;9789241500470. Google Scholar


2. Minakawa N, Omukunda E, Zhou G, Githeko A, Yan G. Malaria vector productivity in relation to the highland environment in Kenya. The American Journal of Tropical Medicine and Hygiene. 2006;75(3):448-453. PubMed | Google Scholar


3. Hochman S, Kim K. The impact of HIV and Malaria co-infection: what is known and suggested venues for further study. Interdiscip Perspect Infect Dis. 2009;2009:617954. PubMed | Google Scholar


4. Guerra CA, Gikandi PW, Tatem AJ, Noor AM, Smith DL, Hay SI et al. The limits and intensity of Plasmodium falciparum transmission: implications for malaria control and elimination worldwide. PLoS Med. 2008;5(2):e38. PubMed | Google Scholar


5. Anstey NM, Russell B, Yeo TW. The pathophysiology of vivax malaria. Trends Parasitol. 2009;25(5):220-227. PubMed | Google Scholar


6. Wilson NO, Bythwood T, Solomon W, Jolly P, Yatich N, Jiang Yi et al. Elevated levels of IL-10 and G-CSF associated with asymptomatic malaria in pregnant women. Infectious Diseases in Obstetrics and Gynaecology. 2010;2010(317430):7. PubMed | Google Scholar


7. Laishram DD, Sutton PL, Nanda N, Sharma VL, Sobti RC, Carlton JM et al. The complexities of malaria disease manifestations with a focus on asymptomatic malaria. Malar J. 2012;11:29. PubMed | Google Scholar


8. Ballas, SK. More definitions in Sickle cell disease: Steady state v Base Line data. Am J Haematol. 2012;87(3):338. PubMed | Google Scholar


9. Alves FP, Gil LHS, Marrelli MT, Ribolla PEM, Camargo EP, Da Silva HP. Asymptomatic Carriers of Plasmodium Spp. as infection Source for Malaria vector Mosquitoes in the Brazilian Amazon. J Med Entomol. 2005; 42(5):777-779. PubMed | Google Scholar


10. Akanmori BD, Kurtzhals JA, Goka BQ, Adabayeri V, Ofori MF, Nkrumah FK et al. Distinct patterns of cytokine regulation in discrete clinical forms of Plasmodium falciparum malaria. Eur Cytokine Netw. 2000; 11(1): 113-118. PubMed | Google Scholar


11. Rodrigues-da-Silva RN, daCosta Lima- Junior J, Fonseca e Fonseca BP, Zuquim Antas PR, Baldez A, Storer FL et al. Alterations in cytokines and haematological parameters during acute and convalescent phases of Plasmodium falciparum and Plasmodim vivax infections. Mem Inst Oswaldo Cruz. 2014;109(2):154-162. PubMed | Google Scholar


12. Baptista JL, Vanham G, Wery M, van Marck E. Cytokine levels during mild and cerebral falciparum malaria in children living in a mesoendemic area. Tropical Medicine and International Health. 1997;2(7):673-679. PubMed | Google Scholar


13. Couper KN, Blount DG, Riley EM. IL-10: The master regulator of immunity to infection. J Immunol. 2008;180(9):5771-5777. PubMed | Google Scholar


14. Langhorne J, Ndungu FM, Sponaas AM, Marsh K. Immunity to malaria: more questions than answers. Nat Immunol. 2008;9(7):725-732. PubMed | Google Scholar


15. Andrade BB, Reis-Filho A, Souza-Neto SM, Clarencio J, Camargo LM, Barral A et al. Severe Plasmodium vivax malaria exhibits marked inflammatory imbalance. Malar J. 2010;9:13. PubMed | Google Scholar


16. Bottius E, Guanzirolli A, Trape J, Regier C, Konate L, Druilhe P. Malaria: even more chronic in nature than previously thought; Evidence for subpatent parasitaemia detectable by the polymerase chain reaction. Trans R Soc Trop Med Hyg. 1996;90(1):15-19. PubMed | Google Scholar


17. Golassa L, Enweji N, Erko B, Aseffa A, Swedberg G. Detection of a substantial number of sub-microscopic Plasmodium falciparum infections by polymerase chain reaction: a potential threat to malaria control in Ethiopia. Malar J. 2013;12:352. PubMed | Google Scholar


18. Trape JF, Zoulani A, Quinet MC. Assessment of the incidence and prevalence of clinical malaria in semi-immune children exposed to intense and perennial transmission. Am J Epidemiol. 1987;126(2):193-201. PubMed | Google Scholar


19. Williams TN, Obaro SK. Sickle cell disease and malaria morbidity: a tale with two tails. Trends Parasitol. 2011;27(7):315-320. PubMed | Google Scholar


20. Kotila R, Okesola A, Makanjuola O. Asymptomatic malaria parasitaemia in Sickle disease patients: How effective is chemoprophylaxis. J Vect Borne Dis. 2007;44(1):52-55. PubMed | Google Scholar


21. Salawu L, Orimolade EA, Durosimi MA. Immuno-Haematological characteristics of Nigerian Sickle Cell disease patients in Asymptomatic steady state. Eur J Gen Med. 2009; 6(3):170-174. Google Scholar


22. May J, Evans JA, Timmann C, Ehmen C, Busch W, Thye T, Agbenyega T, Horstmann RD. Hemoglobin variants and disease manifestations in severe falciparum malaria. JAMA. 2007; 297(20):2220-2226. PubMed | Google Scholar


23. Worku L, Damtie D, Endris M, Getie S, Aemero M. Asymptomatic Malaria and Associated Risk Factors among School Children in Sanja Town, Northwest Ethiopia. International Scholarly Research Notices. 2014; 2014(303269):6. PubMed | Google Scholar


24. Santana-Morales MA, Afonso-Lehmann RN, Quispe MA, Reyes F, Berzosa P, Benito A et al. Microscopy and molecular biology for the diagnosis and evaluation of malaria in a hospital in a rural area of Ethiopia. Malar J. 2012;11:199. PubMed | Google Scholar


25. Singh B, Bobogara A, Cox-Singh J, Snounou G, Abdullah MS, Rahman HA. A genus- and species-specific nested polymerase chain reaction malaria detection assay for epidemiologic studies. Am J Trop Med Hyg. 1993;60(4): 687-692. PubMed | Google Scholar


26. Modell B, Darlison M. Global epidemiology of haemoglobin disorders and derived service indicators. Bull World Health Organ. 2008;86(6):480-487. PubMed | Google Scholar


27. Serjeant GR, Ndugwa CM. Sickle cell disease in Uganda: a time for action. East Afr Med J. 2003;80(7):384-387. PubMed | Google Scholar


28. Serjeant GR. Mortality from sickle cell disease in Africa. BMJ. 2005;330(7489):432-433. PubMed | Google Scholar


29. McAuley, CF, Webb C, Makani J, Macharia A, Uyoga S, Opi DH et al. High mortality from Plasmodium falciparum malaria in children living with sickle cell anaemia on the coast of Kenya. Blood. 2010;116(10):1663-1668. PubMed | Google Scholar


30. Makani J, Cox SE, Soka D, Komba AN, Orua J. Mortality in Sickle Cell Anaemia in Africa: A Prospective Cohort Study in Tanzania. Plos One. 2011;6(2): e14699. PubMed | Google Scholar


31. Geerligs PDP, Brabin BJ, Eggelte TA. Analysis of the effects of malaria chemoprophylaxis in children on haematological responses, morbidity and mortality. Bull World Health Organ. 2003;81(3):205-216. PubMed | Google Scholar


32. Lengeler C. Insecticide-treated bed nets and curtains for preventing malaria. Cochrane Database Syst. Rev. 2004;2:CD000363. PubMed


33. Dhiman S, Goswami D, Rabha B, Yadav, K, Chattopadhyay P, Veer V. Absence of asymptomatic malaria in a cohort of 133 individuals in a malaria endemic area of Assam, India. BMC Public Health. 2015;15:919. PubMed | Google Scholar


34. Doni NY, Zeyrek FY, Seyrek A. Detection of Plasmodium using flter paper and nested PCR for patients with malaria in Sanliurfa, in Turkey. Malar J. 2016;15(1):299. PubMed | Google Scholar


35. Strom GEA, Tellevik MG, Fataki M, Langeland N, Blomberg B. No asymptomatic malaria parasitaemia found among 108 young children at one health facility in Dar es Salaam, Tanzania. Malar J. 2013;12:417. PubMed | Google Scholar


36. Ganguly S, Saha P, Guha SK, Biswas A, Das S, Kundu PK et al. High prevalence of asymptomatic malaria in a Tribal population in Eastern India. J Clin Microbiol. 2013;51(5): 1439-1444. PubMed | Google Scholar


37. Luty AJ, Perkins DJ, Lell B, Schmidt-Ott R, Lehman LG, Luckner D et al. Low Interleukin-12 activity in severe Plasmodium falciparum malaria. Infect Immun. 2000;68(7):3909-3915. PubMed | Google Scholar


38. Guiyedi V, Becavin C, Herbert F, Gray J, Cazenave P, Kombila M et al. Asymptomatic Plasmodium falciparum infection in children with increased auto-antibody production, high IL-10 plasma levels and antibodies to merozoite surface protein 3. Malar J. 2015;14:162. PubMed | Google Scholar


39. Hugosson E, Montgomery SM, Premji Z, Troye-Blomberg M, Bjorkman A. Higher IL-10 levels are associated with less clearance of Plasmodium falciparum parasites. Parasite Immunology. 2004; 26(3):111-117. PubMed | Google Scholar