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Fibrinogen and C-Reactive Protein Significance in Children Infected by Plasmodium falciparum Species in Enugu, Enugu State, Nigeria

Malaria accounts for a considerable mortality and morbidity rate with children bearing the greatest burden. The study investigated fibrinogen and C-reactive protein (CRP) value alterations in children infected by Plasmodium falciparum (P. falciparum) species. A case control study with a total of Original Research Article Ogbonna et al.; JPRI, 33(15): 1-8, 2021; Article no.JPRI.66634 2 ninety-five microscopically confirmed P. falciparum malaria infected children and fifty apparently healthy age and gender matched controls from Enugu State University Teaching Hospital, Parklane, Wesley Specialist Hospital and Akpugo Community Health Centre, Enugu were recruited for the study. Fibrinogen level was determined by clauss clotting time method using sodium citrated plasma. Giemsa stained thick and thin blood film was used for parasite identification and calculation of parasite density. Serum CRP values was determined by immunoturbidimetric method. Fibrinogen levels were significantly increased (p < 0.05) in P. falciparum infected children (324.03 + 59.87) mg/dl as compared to the control (224.74 + 34.88) mg/dl. Parasite density showed a weak positive correlation between fibrinogen (p < 0.01, r = 0.461) and CRP (p < 0.01, r = 0.232). CRP was significantly increased (p < 0.05) in P. falciparum malaria infected children (21.52 + 35.59) mg/l as compared to the control (2.43 + 0.97) mg/l. In conclusion, P. falciparum malaria infection demonstrated a significant impact on fibrinogen and CRP.


INTRODUCTION
Malaria still remains a global burden with its complication in childhood posing a great threat to public health [1,2]. In hyperendemic and holoendemic malarial areas in Sub-Saharan Africa including Nigeria, it is the most widespread and life threatening human protozoal infection [3]. It is certainly one of the diseases exerting a huge economic burden on families, communities and the country at large [4,5]. While children under the age of five and pregnant women are particularly vulnerable, almost the entire population of Nigeria is at risk of contracting malaria [6]. Malaria accounts for an estimated 212 million cases worldwide and 429,000 deaths mostly in the African region [7].
Malaria is primarily caused by parasites transmitted from one person to another through the bites of infected female anopheles mosquitoes [8]. The protozoan parasites of genus plasmodium that infect humans include; Plasmodium falciparum, Plasmodium ovale, Plasmodiun vivax and Plasmodium malariae with P. falciparum causing more deadly infection [9,10]. Plasmodium falciparum infections affect single and multiple organs resulting to organ failure, severe anaemia, cerebral malaria, coma and consequently death [11]. Other ways include; contact with infected blood, from mother to foetus before/during delivery [12], congenitally acquired disease, sharing of contaminated needles and organ transplantation [13].
Haemostatic changes which could be as a result of fibrinolytic activity, is one of the risk factors associated with severity of malaria infection [14]. Fibrinogen reduces P. falciparum erythrocyte membrane protein 1 (PfEMP-I) binding to intracellular adhesion molecule 1 (ICAM-1) hindering sequestration thus affecting malaria pathogenesis [15]. In P. falciparum malaria infection, the reduced fibrinolytic activity and moderately high fibrinogen level are contributing factors to the possibility of thromboembolic process occurring in children thus increasing the risk of mortality [14]. Severe haemostatic abnormalities in malaria are manifested in thrombocytopenia, decreased activity of coagulation and disseminated intravascular coagulation [16].
Parasite density has been shown from previous studies to have positive correlation with the severity of malaria infection [17]. The degree of parasitaemia also correlates with mortality rate. Hyperparasitemia is defined as > 2% of infected erythrocyte or more than 100,000 parasite/µl in low intensity transmission arrears or > 5% of infected erythrocyte or more than 250,000 parasite/µl in areas of high stable malaria transmission intensity [18]. In severe P. falciparum malaria, hyperparasitemia has been indicated in anaemia, thrombocytopenia, cerebral malaria and disseminated intravascular coagulation [19].
C-reactive protein is a positive acute phase marker of inflammation induced by the release of inflammatory cytokines (IL-6) secreted during entry of parasite into the erythrocytes [1]. It is an important immunologic indicator for active malarial episode and studies have shown that CRP levels correlate with parasite burden especially in P. falciparum malaria infection [20]. CRP is secreted in increased amount within six hours of an acute inflammatory stimulus and after effective treatment or removal of the inflammatory stimulus, the level falls [21]. It has a pathogenic role of binding to infected red cells, accelerating phagocytosis, activating the complement pathway and detoxification of substance released from the damaged tissue [22].
The study aimed to investigate the fibrinogen and C-reactive protein value alterations in children infected by P. falciparum species in Enugu, Enugu State, Nigeria.

Study Area
The study was done in Enugu State, Nigeria.

Subjects
A total of 145 patients were studied. 95 were children infected with P. falciparum malaria between the ages of 1-10 years and 50 were apparently healthy children (controls), sex and age matched. The patients were not on antimalarial treatment as at the time of sample collection and had no associated infection on clinical examination.

Sample Size
The sample size, n for the study will be calculated using the formula below by Araoye. n= the desired sample size when the population is more than 10,000 Z = standard deviation usually set at 1.96, which corresponds to 95% confidence interval P = the proportion in the target population estimated to have a particular characteristics. (Prevalence and severity of malaria parasitaemia among children requiring emergency blood transfusion in tertiary hospital in Imo state, Nigeria) by Austin et al. [23]

Study Design
This was a case control study focused on children between 1-10years of age at Enugu State University Teaching Hospital, Parklane, Wesley Specialist Hospital, Enugu and Akpugo community Health Centre. This study ran from April, 2018 to September, 2018 covering the wet season when the rate of mosquito bite is at its peak. The patients were recruited consecutively and prospectively.

Inclusion criteria
Inclusive subjects were male and female children between the ages of 1-10years who were not on antimalarial drugs. Only patients positive for P. falciparum malaria infection were included in the study.

Exclusion criteria
Children above 10 years of age and those who refused to give their consent were excluded from this study. Children with sickle cell disease were also excluded from this study because malaria parasite does not grow well in sickle cells.

Sample Collection
Blood sample (6.0ml) was collected by venepuncture under asceptic conditions, 2ml of the blood sample was transferred into the ethylene-diamine-tetra-acetic-acid (EDTA) container for malaria parasite test, 2ml was transferred into plain tube for biochemical analysis and 2ml was transferred to trisodium citrate container for coagulation studies.

Parasitologic Examination of Blood Samples
Giemsa stained thick and thin blood films were prepared for each sample and parasitaemia evaluated per microliter of blood using the thick film preparation according to standard method described by World Health Organisation assuming a mean total leukocyte count of 8000/µl of blood. This was done using Olympus binocular microscope. A slide was considered negative when no malaria parasite was seen in 100 high power fields.

C-Reactive Protein Assay
Quantitative determination of serum CRP was performed by immunoturbidimetric method using a biochemical analyzer, Eppendorf Biophotometer plus at 340nm. The reagent was from Fortress diagnostics (product code: BXC0384, United Kingdom).

Statistical Analysis
Data were subjected to descriptive statistics and analyzed using analysis of variance and student's T-test. The probability value less than 0.05 were considered statistically significant.

Characteristics of Study Population
A total of 145 children (positive for P. falciparum malaria infection and controls) were sampled from hospitals in Enugu, Enugu State. It included 74 (51%) males and 71 (49%) females with male to female ratio of 1.04:1. The mean age of the infected males and females and that of the control group were similar (1-10years) as shown in Table 1. All the infected patients were of P. falciparum malaria parasite specie and no patient with dual infection was included in this study.

Effect of Malaria on Fibrinogen and C Reactive Protein (Crp)
The comparison of fibrinogen and CRP between the P. falciparum malaria infected children and the controls are shown in

Relationship Between Parasite Density and Parameters
A correlation between parasite density with fibrinogen and CRP is shown in the Table 3. respectively and Fig. (1 and 2). A weak positive correlation was found between parasite density and fibrinogen level (p < 0.01, r = 0.461) CRP (p < 0.01, r = 0.232).

DISCUSSION
The observed significant increase in fibrinogen level could be due to increased secretion of the hepatic cells caused by the inflammatory mediators released in malarial infection and also increased activation of the coagulation cascade. A study by Omoigberale [14] reported a significantly increased fibrinogen levels in malaria infected patients compared to the controls. He further stated that "children with malaria infection had a decreased fibrinolytic activity and a proportionately high fibrinogen level". Few published research are done on the effect of malaria on fibrinogen level. Disseminated intravascular coagulation, a consumptive coagulopathy arising due to increased turnover of the coagulation cascade is characterized with low fibrinogen level [24].
The observed weak positive correlation between parasite density some parameters (fibrinogen and CRP) indicates that increased malaria parasitemia increases inflammatory response and consequently increase fibrinogen level. Studies have shown correlation between parasitic density and severity of malaria infection [17]. In severe P. falciparum malaria, hyperparasitemia indicates poor prognosis of cerebral malaria.
According to WHO, hyperparasitemia is defined as > 2% of infected erythrocyte or more than 100,000 parasite/µl in low intensity transmission arrears or > 5% of infected erythrocyte or more than 250,000 parasite/µl in areas of high stable malaria transmission intensity [18]. High P. falciparum parasitamia has been indicated in anaemia due to excessive destruction of parasitized red cells and thrombocytopenia due to increased platelet consumption [19].
The significant increase in CRP might probably be due to increased hepatic stimulations and production. This higher level of mean serum CRP in infected patients further reinforces malarial effect on the induction of CRP. C-reactive protein is markedly increased in acute inflammatory events and the degree of increase reflects the severity of the inflammatory response in malaria infection [25]. Various authors have documented an association of high circulating levels of inflammatory cytokines such as TNF, IL-1 and IL-6 to severe malaria. The significantly increased serum CRP seen in this

Fig. 1. Correlation between parasite density and fibrinogen
study is in accordance with the research by Utuk et al. [1] who reported higher level of CRP in children infected with P. falciparum malaria. Paul et al. [26] in their study found that CRP levels were significantly increased in malaria infected patients but found no significant difference between P. falciparum and P. vivax case. However, Agrawal et al. [21] found mean CRP to be higher in plasma of P. falciparum infected patients compared to P. vivax infected patients.  (2-tailed) .000 N 145 145 C-reactive protein (mg/L) Pearson Correlation .482 ** 1 Sig. (2-tailed) .000 N 145 145 **. Correlation is significant at the 0.01 level (2-tailed).

CONCLUSION
In conclusion, P. falciparum malaria infection demonstrated a significant impact on fibrinogen and C-reactive protein. Based on the findings of this study, fibrinogen and CRP might probably be good diagnostic markers of inflammation seen in P. falciparum malaria infection.

CONSENT AND ETHICAL APPROVAL
Ethical clearance was obtained from the Health Research and Ethical committee of the University of Nigeria Teaching Hospital, Ituku-Ozalla, Enugu, Enugu State. Informed consent was obtained from the mothers and caregivers of the children before administration of questionnaire and collection of blood sample.