The Significance of TNF-α, CRP, and Hematological Parameters in the Prediction of Plasmodium Falciparum Parasitemia

Malaria remains a significant public health concern in Sudan, with Plasmodium falciparum (P. falciparum) infection having widespread indirect consequences. Effective identification of infection severity is crucial for preventing complications. This study aimed to assess the predictive value of Tumor Necrosis Factor Alpha (TNF-α), C-reactive protein (CRP), and hematological parameters in P. falciparum parasitemia. The three-month cross-sectional study involved 54 P. falciparum-positive patients at Bashaire Teaching Hospital Khartoum, Sudan, along with an equal number of P. falciparum-negative individuals. Participants underwent Antigen Rapid Diagnostic Test (RDT), TNFα, CRP, and Complete Blood Count (CBC) assessments. The infected group comprised 68.5% male patients, with a mean age of 22.3 years. Significant variations were observed between the infected and uninfected groups in TNFα, CRP, Total White Blood Cells (TWBCs), and neutrophil levels, with infected individuals showing higher values. Conversely, infected individuals had lower hemoglobin, Red Blood Cells (RBCs), Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Hemoglobin Concentration (MCHC), platelets, and lymphocytes. The study revealed a noteworthy association between MCV and parasitemia, while TNFα, CRP, Hb, RBCs, MCH, and MCHC showed no significant correlation. Hematological parameters should be considered in malaria patients.

Introduction

Malaria constitutes a significant global health challenge, resulting in an annual death toll of 1 to 3 million people, particularly affecting regions with tropical climates [1], [2]. Urgent and decisive interventions are imperative to mitigate the impact of malaria on public health. Timely and precise diagnosis, as advocated by the World Health Organization (WHO), is paramount in lowering the disease’s morbidity and mortality rates [2]. Of all Plasmodium species capable of infecting humans, P. falciparum stands out due to its association with severe and often fatal complications, including cerebral malaria, acute renal failure, acute malarial hepatitis, hypoglycemia, hyperpyrexia, and other serious conditions [3]. The identification of secondary complications such as hematological abnormalities, involving disruptions to red blood cells, white blood cells, platelets, and elevated C-reactive protein (CRP) levels signifies the presence of inflammation in malaria cases [4], [5]. Thrombocytopenia, a common occurrence in malaria, stems from peripheral destruction, heightened platelet sequestration in the spleen, and consumption linked to disseminated intravascular coagulation [6]. Immune responses mediated by cytokines like TNFα and CRP are pivotal in the disease’s progression, revealing insights into disease severity [7]. This research endeavours to assess the prognostic value of TNFα, CRP, and haematological parameters in predicting parasitemia within P. falciparum infections.

Materials and Methods

Study Population

A cross-sectional study was conducted between January 2018 and July 2019 at Bashaire Teaching Hospital, Khartoum, Sudan involving 54 patients diagnosed with P. falciprum malaria. An equal number of individuals who tested negative for P. falciparum were also included in the study. Exclusion criteria for this study included patients under 18 years old and those with chronic diseases such as hypertension, cardiac disease, and diabetes mellitus [8].

Lab Investigations

All participants provided two blood samples (3 ml each), one in Ethylenediamine tetra-acetic acid (EDTA) for RDT detection of P. falciparum, a thick blood smear preparation for microscope examination, and a CBC. The second was in a plain container for measuring CRP and TNFα. RDT kits were obtained from RightSign® (Hangzhou Biotest Biotic CO., LTD). CBC was performed on a Mindary BC-3200 auto haematological analyzer (Shenzhen, Mindray-biomedical Co., Ltd., China). The serum was separated by centrifugation at 2000 rpm for 5 minutes and kept at −20 °C.

Rapid Diagnostic Test (RDT)

The malaria P. falciparum RDT cassette is a swift quantitative, membrane-based, chromatographic immunoassay designed for the identification of the P. falciparum antigen in whole blood. The membrane comes pre-coated with specific antibodies such as anti-histidine-rich protein II (HRP-II) and anti-plasmodium Lactate Dehydrogenase (pLDH) antibodies. Upon contact with whole blood, the dye conjugate, which is pre-coated on the test cassette, initiates a reaction. This mixture then moves upwards through the membrane via capillary action. It subsequently interacts with the anti-HRP-II antibodies on the membrane at the P. falciparum test line region and with the anti-PLDH antibodies at the pan line region. In cases where the specimen contains HRP-II, PLDH, or both, the appearance of a coloured line in the P. falciparum line region, the pan line region, or both confirm the presence of the respective antigens. Additionally, for procedural control, the presence of a coloured line in the control line region is imperative, indicating the correct volume of specimen has been added, and proper membrane wicking has taken place.

Thick Blood Smear/Parasite Densities

For the detection of parasitemia, a thick-smear technique was used, with the standard Giemsa technique according to World Health Organization (WHO) criteria [9].

Blood films were examined microscopically using 100 oil immersion. Plus, a sign scheme was used to report parasite numbers Table I [10], [11].

Table I. The Parasite Density in Relation to Parasite Count

Group	Parasite density	Parasite count per filed/s	Cross
Group I	1−10	Per 100 high power fields	+
Group II	11−100	Per 100 high power fields	++
Group III	1−10	In every one high power fields	+++
Group IV (high parasitemia)	More than 10	In every one high power fields	++++

C-Reactive Protein Measurement

The CRP levels in the serum were measured using the Nyco-Card Reder II system, which employs a sandwich-form immunometric assay. To conduct this procedure, a diluted sample was administered to a membrane that had been coated with immobilized CRP-specific monoclonal antibodies. The antibodies captured the CRP as the sample flowed through the membrane, after which the trapped CRP was bound by the gold-antibody conjugate that was added in a sandwich-type reaction. Washing of the solution was utilized to remove any unbound conjugate from the membrane, with a paper layer placed underneath the membrane to absorb any remaining liquid. The membrane appears red-brown in the presence of a pathological level of CRP, with the colour intensity of the membrane being proportional to the CRP concentration of the sample. The Nyco-Card Reader II device was used to measure the colour intensity quantitatively. The monoclonal antibodies utilized in the assay were specific to human CRP, with no other human blood components having been found to cross-react with CRP in the Nyco-Card CRP single-test system. Analytical specificity was also verified with accuracy using the mentioned monoclonal antibodies.

TNFα Measurement

TNFα level was measured in the serum using a sandwich Enzyme-Linked immunosorbent assay technique (ELISA). The assay was performed according to the manufacturer protocol (Biolegend® Human TNFα ELISA MAX). The plate includes a stander curve. Absorbance was read against a blank at 450 nm using a microliter ELISA absorbance reader (Infinite F50 Tecan Group Ltd., Mannedrof, Switzerland).

Statistics Analysis

The Statistical Package for Social Sciences (SPSS) version 19 was utilized for data analysis. Frequency and per cent were used to describe qualitative data, while quantitative data was expressed as mean ± standard deviation. The Chi-square test for independence was performed to assess the relationship between qualitative nominal variables, and the Pearson correlation test was utilized for quantitative variables. For two groups, the independent sample test was used to determine differences, while ANOVA was used for three groups.

Ethical Consideration

Verbal informed consent was taken from all participants.

Results

The majority of the malaria-infected group were male (68.5%). The mean age of malaria malaria-infected group was 22.3 years Approximately 18.5% had hyperparasitemia (parasite density was ++++) Table II and Fig. 1. ELISA results showed a significant elevation of TNFα, CRP, TWBCs, and neutrophils, in a malaria-infected group compared to the non-infected group. Hb, RBCs, MCV, MCH, MCHC, platelets and lymphocytes were significantly lower in the malaria-infected group compared to the non-infected group Table III. No significant association was found between parasite density with TNFα, CRP, Hb, RBCs, MCH, and MCHC. MCV was significantly associated with parasite density Table IV.

Table II. Demographic Variables of Malaria Infected Group (Smear Positive) and Non-Infected Group (Smear Negative)

Variables	Smear positive (n = 54)	Smear negative (n = 54)
Gender:
Male	37 (68.5%)	38 (70.4%)
Female	17 (31.5%	16 (29.6%)
Mean age (years)	22.3	20.9

Fig. 1. Degree of parasitemia by crosses among parasitemic (smear positive) group.

Table III. TNF α, CRP, and Heamatological Parameters Among Malaria Infected (Smear Positive) and Non-Infected (Smera Negative) Groups

Parameters	Smear positive (n = 54)		Smear negative (n = 54)		p-value
	Mean	Std. deviation	Mean	Std. deviation
RBCs (×106/µL)	4.51	0.65	5.16	0.48	0.000
MCV (fl)	80.44	6.58	90.04	3.89	0.000
MCH (pg)	26.61	2.76	28.08	1.41	0.002
MCHC (g/dl)	33.18	1.53	30.89	1.05	0.000
HCT %	37.02	6.61	46.02	4.33	0.000
Hb (g/dl)	12.32	1.93	14.57	1.17	0.000
PLTs (×103)	116.57	66.01	291.02	79.43	0.000
TWBCs (×103/µL)	6.94	2.93	5.77	1.43	0.003
Neutrophils (×103)	5.25	2.48	2.95	1.19	0.000
Lymphocytes (×103)	1.09	0.69	1.59	0.73	0.000
TNF α (pg/ml)	27.65	49.52	3.72	0.71	0.001
CRP (mg/L)	51.70	29.6	5.91	1.14	0.000

Table IV. Correlation of TNFα, CRP And Heamatological Profiles With Parasit Density

Parameters	Degree of parasitemia by cross				p-value
	Group I (+)	Group II (++)	Group III (+++)	Group IV (++++)
RBCs (×106/µL)	4.6	4.5	4.4	4.3	0.63
MCV (fl)	78.6	80.7	85.5	77.8	0.02
MCH (pg/cell)	26.0	26.7	28.5	25.4	0.05
MCHC (g/dl)	33.0	33.2	33.3	33.1	0.972
Hb (g/dl)	12.2	12.1	12.7	12.3	0.87
HCT %	36.86	36.5	38.31	36.63	0.928
PLTs (×103)	145.00	105.0	83.2	121.3	0.09
TWBCs (×103/µL)	6.34	6.5	6.51	9.1	0.077
Neutrophils (×103)	4.7	4.78	5.2	6.9	0.113
Lymphocytes (×103)	1.02	1.05	0.99	1.3	0.540
TNFα (pg/ml)	21.76	40.76	17.0	26.0	0.604
CRP (mg/L)	37.35	53.47	64.10	60.70	0.077

Discussion

Hematological abnormalities are commonly observed in malaria infections, with P. falciparum being the most affected form [12]. Our study reports significant changes in various blood parameters among malaria-infected groups, including a marked decrease in RBC counts, haemoglobin, RBC indices (MCH, MCHC, and HCT) and thrombocytopenia which is consistent with previous research findings [12], [13]. Similar to previous findings [13], our study observed leukocytosis and a rise in neutrophil count in the infected group. However, contradictory results have been reported in RBC indices and TWBCs [14]. Additionally, lymphopenia was observed, which coincides with Muwonge et al.’s study [14]. However, no significant association was found between TWBC count, neutrophils, Hb, RBCs, HCT, MCHC, and MCH with parasite density. Contrary to our findings, Lima-Junior et al. reported a significant association between neutrophils and parasite density [15]. The present study demonstrates a significant association between MCV and parasite density which is harmonious with Kotepui et al.’s findings [13].

In contrast to the previous study [6], our findings demonstrate no significant correlation between platelet count and parasite density. Furthermore, our results indicate a statistically significant elevation of TNF-α in the infected group compared to the non-infected group, consistent with previous research [16]. However, we did not observe a significant association between high levels of TNF-α and parasite density. It is worth noting that, in contrast to our findings, TNF-α levels were strongly correlated with parasite density among children with severe falciparum malaria in endemic areas of Sudan [17], [18].

CRP levels, a biomarker for malaria detection and monitoring malaria severity [19], were observed to be significantly elevated in our study. The absence of a significant correlation between high CRP levels and parasite density is congruent with other previous studies [20], [21].

Conclusions

Parasitemia was negatively associated with TNFα and CRP, and positively affected MCV. Hematological parameters should be considered in malaria patients.

Recommendations

Further studies are needed to examine the effect of parasitemia on TNFα and CRP. Reviewing the investigation management protocol of malaria in Sudan in a continuous manner.