Evaluation of Immunophenotypic Alterations of Peripheral Blood Lymphocytes and their Sub-Sets in Uncomplicated P. Falciparum Infection

doi:10.21203/rs.3.rs-2845374/v1

Background: Malaria is a life-threatening parasitic disease typically transmitted through the bite of an infected Anopheles mosquito. There is ample evidence showing the potential of malaria infection to affect the counts of lymphocyte subpopulations in the peripheral blood, but the extent of alteration might not be consistent in all geographical locations, due to several local factors. Although Ghana is among the malaria endemic countries, there is currently no available data on the level of alterations that occur in the counts of lymphocyte subpopulations during Plasmodium falciparum malaria infection among adults.

Aim: The study was to determine the immunophenotypic alterations in the level of peripheral blood lymphocytes and their subsets in adults with uncomplicated P. falciparum malaria infection and apparently healthy participants.

Methods: The study was a cross-sectional comparative study conducted in two municipalities of the Volta region of Ghana. Blood samples were collected from study participants and taken through serology (P. falciparum/Pan Rapid Diagnostic Kits), microscopy (Thick and thin blood films) and Haematological (Flow cytometric and Full blood count) analysis.

Results: A total of 414 participants, comprising 214 patients with malaria (cases) and 200 apparently healthy individuals (controls) were recruited into this study. Parasite density of the malaria patients ranged from 75/µL to 84,364/µL, with a mean of 3,520/µL. It was also observed that the total lymphocytes slightly decreased in the P. falciparum infected individuals (Mean ± SD: 2.08 ± 4.93 × 10⁹/L) compared to the control group (Mean ± SD: 2.47 ± 0.80 × 10⁹/L). Again, there was a significant medium positive correlation between parasite density and haematocrit levels (r= 0.321, p < 0.001). Apart from CD45+ T-cells, more people in the control group had normal values for the lymphocyte subsets measured compared to the cases.

Conclusions: From the results obtained, there was high parasite density among the malaria patients suggestive of high intensity of infection in the case group. The cases again showed considerable haematological alterations in lymphocyte sub-sets and the parasite density appeared to be strongly associated with CD4+ T-cell reduction. Also, the parasite density significantly associated with decreasing haematocrit levels.

Haematological

Immunophenotypic

Parasite density

P. falciparum

Lymphocytes and Malaria

Malaria is a life-threatening parasitic disease typically transmitted through the bite of an infected Anopheles mosquito which carries the erythrocytic protozoan of the phylum apicomplexa and genus Plasmodium [1]. Malaria in man is caused by five main species of Plasmodium, namely: P. falciparum, P. vivax, P. ovale, P. malariae and P. knowlesi [1]. Among these species, P. falciparum and P. vivax pose the greatest threat to humans, but P. falciparum is considered the deadliest [2, 3]. The World Health Organization (WHO) defines malaria as uncomplicated when symptoms are present, but there are no clinical or laboratory signs to indicate severity or vital organ dysfunction [4]. It is refreshing to note that the vast majority of malarial infections cause uncomplicated malaria, with only approximately 1–2% of these episodes becoming severe [5].

Malaria parasites have been identified to disrupt the normal profile of immune cells in the peripheral blood. For example, there have been reports on alterations in the proportions of total leukocytes, total lymphocytes, Natural Killer cells, αβ and γδ T-cell, and B-cell counts during Plasmodium falciparum and Plasmodium vivax infections [6, 7]. Severe P. falciparum malaria is also associated with increased plasma levels of a wide range of cytokines and markers of T cell activation and endothelial inflammation [8, 9]. It is also associated with T cell lymphopenia and impaired ability of peripheral blood T cells to produce some cytokines in vitro [9].

Furthermore, it has been postulated that, CD8 + T cells and the cytokines IFN-γ and TNF provide protection against pre-erythrocytic Plasmodium within hepatocytes, whereas CD4 + T cells restrict growth of Plasmodium parasites in erythrocytes through secretion of cytokines, activation of macrophages and general direction of humoral immunity [10, 11]. The contribution of regulatory T cells in malaria infection has also been demonstrated, suggesting that the balance between pro-and anti-inflammatory cytokines is needed to monitor changes related to malaria [12, 13]. Although Ghana is among malaria endemic countries, there is currently no available data on the level of alterations that occur in lymphocyte subpopulations during P. falciparum malaria infection among adults. Thus, this study, it is believed will fill the gap and inform the implementation of special steps when dealing with malaria patients, such as enumeration of peripheral lymphocyte cells for diagnostic purposes and to evaluate the immune status of malaria patients.

Demographic and social features of the study participants

In total, four hundred and fourteen participants were recruited into this study, comprising 214 patients with malaria (cases) and 200 apparently healthy individuals (controls). The mean ages of the malaria-infected and control groups were 36.6 and 31.6 years respectively. In the former, a greater proportion of the study participants were females (57.9%, n = 124), whereas in the latter, males dominated (59.5%, n = 119). Again, the data shows that 76.6% of the cases have stayed in the region for 2–10 years, followed by 18.7% who have stayed for 11–20 years, whilst the control group gave 75% and 15%, for these years respectively. Regarding the employment status of the participants, the cases reported 55.1% for employed, 42.4% for unemployed and 1.4% for retired whilst the control group recorded 51%, 48% and 1% for same parameters respectively. Also, data on the educational background revealed that 28% of the malaria cases had attained basic education compared to 30.5% of the control and 43.9% with secondary education compared to 28% for the control group. Again, among the cases, 69.2% were known to have their houses close to stagnant waters whilst the control group recorded 22%. Also, with regards to the use of Insecticide Treated Nets (INTs), 49.1% said Yes to whether they use ITNs whilst 50.9% said No, with the control group recording 93.5% and 6.5% for Yes and No respectively (Table 1).

Table 1

Demographic features of the study participants
Features	Mean ± SD	Malaria cases(N = 214)		Control (N = 200)
Features		Number	%	Number	%
Age
Cases	31.58 ± 12.27
Controls	36.6 ± 10.06
Gender
Male		90	42.1	119	59.5
Female		124	57.9	81	40.5
Years of stay in the region
2–10		164	76.6	150	75
11–20		40	18.7	30	15
21–30		7	3.27	15	7.5
31–40		1	0.47	5	2.5
Employment status
Employed		118	55.1	102	51
Unemployed		91	42.5	96	48
Retired		3	1.4	2	1
Education
Basic		60	28.0	61	30.5
Secondary		94	43.9	56	28
Tertiary		46	21.5	74	37
Uneducated		12	5.6	9	4.5
Closeness of abode to stagnant water
Yes		148	69.2	44	22
No		64	29.9	156	78
Use of ITN
Yes		105	49.1	187	93.5
No		109	50.9	13	6.5

Enumeration of parasite density of the cases.

When the parasite density was analyzed among the cases, they ranged from 75/µL to 84,364/µL, with a mean of 3,520/µL and most (129/214, 60.3%) had parasite density within x10², whilst 28.5% had within x10³. Meanwhile, none (0%) of the patients recorded parasite density within single digit number (Fig. 1).

Insert Fig. 1

General Haematological Parameters Among The Study Participants

Regarding haematological parameters among the two study groups, it was observed that the total lymphocytes were slightly decreased among the cases (Mean ± SD: 2.08 ± 4.93 × 10⁹/L) compared to the control group (Mean ± SD: 2.47 ± 0.80 × 10⁹/L). Other notable decreases included haemoglobin (13.61 ± 1.22 g/dl vs 11.53 ± 2.03 g/dl), haematocrit (1.27 ± 8.92 vs 0.33 ± 0.06), mean corpuscular volume (170.00 ± 860.90 fL vs 82.61 ± 9.03 fL), mean corpuscular haemoglobin (38.06 ± 48.21 pg vs 28.18 ± 2.92 pg) and platelets (203.64 ± 78.74 × 109/L vs 145.45 ± 70.04 × 109/L) (Table 2).

Table 2

**General Haematological parameters among the study participants**
Haematological Component	Mean ± SD
	Control group	Malaria cases group
Red blood cells	4.50 ± 0.51 × 10¹²/L	4.07 ± 0.72 × 10¹²/L
Haemoglobin	13.61 ± 1.22 g/dl	11.53 ± 2.03 g/dl
White blood cells	6.43 ± 2.26 × 10⁹/L	6.64 ± 4.31 × 10⁹/L
Neutrophils	3.36 ± 2.14 × 10⁹/L	4.20 ± 2.90 × 10⁹/L
Lymphocytes	2.47 ± 0.80 × 10⁹/L	2.08 ± 4.93 × 10⁹/L
Monocytes	0.40 ± 0.18 × 10⁹/L	0.44 ± 0.24 × 10⁹/L
Eosinophils	0.20 ± 0.19 × 10⁹/L	0.10 ± 0.21 × 10⁹/L
Basophils	0.06 ± 0.05 × 10⁹/L	0.05 ± 0.04 × 10⁹/L
Haematocrit	1.27 ± 8.92	0.33 ± 0.06
Mean corpuscular volume	170.00 ± 860.90 fL	82.61 ± 9.03 fL
Mean corpuscular haemoglobin	38.06 ± 48.21 pg	28.18 ± 2.92 pg
Mean corpuscular haemoglobin concentration	180.08 ± 164.97 g/dL	331.37 ± 58.37 g/dL
Red cell distribution-coefficient variation	2.09 ± 10.55	0.16 ± 0.05
Red cell distribution-standard deviation	50.74 ± 47.75 fL	49.41 ± 11.20 fL
Platelets	203.64 ± 78.74 × 10⁹/L	145.45 ± 70.04 × 10⁹/L
Mean platelet volume	10.71 ± 1.02 fL	10.34 ± 1.13 fL
Platelet distribution width	16.05 ± 0.69	16.03 ± 0.57
Plateletcrit	1.90 ± 0.61 mL/L	1.50 ± 0.72 mL/L
Platelet large cell count	57.01 ± 18.02 × 10⁹/L	43.74 ± 22.09 × 10⁹/L
Platelet large cell ratio	0.31 ± 0.07	0.31 ± 0.08
Mean parasite density of the malaria-infected group (cases) = 3520.12 ± 11820.38069

Insert Table 2.

Levels of lymphocyte subsets among the cases.

Levels of lymphocyte subsets, CD3⁺, CD8⁺, CD4⁺ and CD45⁺ were measured among the two study groups based on BD reference rages [14]. The levels were categorized into low, normal and high. For CD3⁺, 93.5% (200/214) of the control group were within the normal range compared to 45.3% (97/214) of the cases. Thirty-seven (37) of the cases representing 17.3% (37/214) had high CD3⁺ values compared to 2.5% (5/200) of the control group. Regarding CD8⁺, 96.5% (193/200) of the control group had normal values, compared to 48.6% (104/214) of the cases. Similar observation was made for CD4⁺ values with more people (136/214) in the cases having low values, compared to the control group (189/200) (Fig. 2). Meanwhile, all the participants in the control group had low CD45⁺ values (Fig. 2).

Insert Fig. 2.

Independent Samples T Test Of The Haematologic Parameters Of The Participants

The independent samples t-test conducted revealed significant differences between the cases and the control group, in terms of the means of several of their haematologic parameters. For instance, the control group had significantly higher mean red blood cell count (mean difference = 0.43, p < 0.001), haemoglobin level (mean difference = 2.08, p < 0.001), mean corpuscular haemoglobin (mean difference = 9.88, p = 0.004), mean red cell distribution-coefficient variation (mean difference = 1.93, p = 0.011), and lower mean corpuscular haemoglobin concentration (mean difference = -151.29, p < 0.001) than the cases. The total white blood cell count as a whole was not significantly different from one group to the other, but specific white cell types – neutrophils (mean difference = -0.83, p = 0.001), eosinophils (mean difference = 0.10, p < 0.001) and basophils (mean difference = 0.01, p = 0.01) significantly differed from one group to the other. Also, the mean differences regarding haematocrit, mean corpuscular volume, and red cell distribution-standard deviation were not statistically significant. Furthermore, the eta squared statistic computed indicated that the magnitudes of these differences ranged between small and large (Table 3). Similarly, results of the Z-ratio computed, highlighted significant differences between the cases and control participants in terms of their lymphocytic parameters (Table 4)

Table 3

Independent samples t test of the haematologic parameters of the participants
Haematologic parameters	t	Eta squared	df	p value	Mean difference	95% CI
Red blood cells	7.02	0.11	384.04	< 0.001	0.43	0.31–0.54
Haemoglobin	12.73	0.31	352.47	< 0.001	2.08	1.76–2.40
White blood cells	-0.64	0.00	326.13	0.525	-0.21	-0.87–0.44
Neutrophils	-3.34	0.03	390.97	0.001	-0.83	-1.32 – -0.34
Lymphocytes	1.15	0.01	225.06	0.250	.39451	-0.28–1.07
Monocytes	-1.70	0.01	399.03	0.091	− .03501	-0.08–0.01
Eosinophils	4.84	0.05	411.51	< 0.001	.10	0.06–0.14
Basophils	2.51	0.02	412	0.012	.01137	0.00–0.02
Haematocrit	1.50	0.01	199.02	0.136	.94484	-0.30–2.19
Mean corpuscular volume	1.44	0.01	199.04	0.152	87.38969	-32.55–207.33
Mean corpuscular haemoglobin	2.89	0.04	200.36	0.004	9.87586	3.14–16.61
Mean corpuscular haemoglobin concentration	-12.27	0.38	245.15	< 0.001	-151.29464	-175.58 – -127.01
Red cell distribution-coefficient variation	2.58	0.03	199.01	0.011	1.92646	0.456–3.39
Red cell distribution-standard deviation	.384	0.00	219.44	0.702	1.32802	-5.50–8.15
Platelets	7.92	0.14	398.52	> 0.001	58.18940	43.75–72.63
Mean platelet volume	3.43	0.03	412	0.001	.36301	0.16–0.57
Platelet distribution width	0.45	0.00	385.11	0.650	.02827	-0.09–0.15
Plateletcrit	6.08	0.08	407.33	> 0.001	.39719	0.27–0.53
Platelet large cell count	6.71	0.10	404.68	> 0.001	13.26668	9.38–17.15
Platelet large cell ratio	0.70	0.00	411.33	0.487	.00507	-0.01–0.02

Table 4

Z score results of the lymphocytic parameters of the study participants
	Control (n = 200)	Malaria cases (n = 214)	Z score	p value
CD3
Low	8 (4%)	80 (37.4%)	-8.297	< 0.0002
Normal	187 (93.5)	97 (45.3%)	10.553	< 0.0002
High	5 (2.5%)	37 (17.3%)	-4.981	< 0.0002
CD8
Low	0 (0%)	52 (24.3%)	-	-
Normal	193 (96.5%)	104 (48.6%)	10.817	< 0.0002
High	7 (3.5%)	58 (27.1%)	-6.597	< 0.0002
CD4
Low	9 (4.5%)	136 (63.8%)	-	-
Normal	189 (94.5%)	51 (23.9%)	-12.986	< 0.0002
High	2 (1%)	26 (12.2%)	-	-
CD45
Low	200 (100%)	104 (48.6%)	-	-
Normal	0	13 (6.1%)	-	-
High	0	97 (45.3%)	-	-

Associations Between Parasite Density And Haematological Parameters

There was a significant medium positive correlation between parasite density and haematocrit levels (r = 0.321, p < 0.001). The coefficient of determination for this association was 0.103, indicating that parasite density and haematocrit levels share 10.3% of their variance. The other parameters were not significantly associated with parasite density (Table 5).

Table 5

Associations between parasite density and specific haematological parameters
Haematological parameters	r	r²	p
White blood cell count	0.129	0.017	0.06
Neutrophil count	0.094	0.009	0.172
Lymphocyte count	0.089	0.008	0.194
Monocyte count	-0.013	0.000	0.852
Eosinophil count	0.006	0.000	0.932
Basophil count	0.045	0.002	0.509
Platelet count	0.088	0.001	0.198
Red blood cell count	-0.050	0.003	0.470
Haemoglobin level	-0.054	0.003	0.429
Haematocrit level	0.321**	0.103**	< 0.001
**Significant at 0.05 alpha level

This study involved a total of four hundred and fourteen participants, comprising two hundred and fourteen patients with malaria referred to as cases and two hundred apparently healthy individuals who served as controls. The mean age of the cases was slightly higher than that of the control group and a greater proportion of the cases were females whereas the controls were dominated by males. Regarding employment status of the participants, the cases reported 55.1% for employed, 42.4% for unemployed and 1.4% for retired whilst the control group recorded 51%, 48% and 1% for employed, unemployed and retired, respectively. Also, on the educational background it was revealed that 28% of the malaria cases had attained basic education compared to 30.5% of the control and 43.9% with secondary education compared to 28% for the control group. In terms of abode, majority of the case group were known to have their houses close to stagnant water whilst the opposite was the case for the control group. Also, with the use of insecticide treated nets (INTs), 49.1% of the cases said Yes to whether they use ITNs whilst 93.5% of the control group said to ITN use.

In determining the degree of malaria infection (parasite density) among the cases, it was observed that their parasite density ranged between 75/µL – 84364/µL, with a mean parasite density of 3,520/µL, indicating generally high parasite count. This finding was similar to a study by Ayoyo (2009) and Jalal et al., (2016) who found a relatively higher parasite density (parasitaemia ≥ 5000/µL) among their study participants [15, 16]. In high-transmission areas such as the current study location, it has been seen that a higher proportion of the population may be diagnosed with parasites on the basis of microscopy findings but may be asymptomatic and asymptomatic parasite densities often extend up to 10 000 parasites/µL [17]. Therefore, it is not surprising that microscopy employed in this study was able to show observation of high intensity of the infection.

Regarding haematological parameters, it was observed that the cases had a significantly lower red blood cell count, haemoglobin level, haematocrit level, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, red cell distribution, platelet counts, mean platelet volume, platelet distribution width, plateletcrit, as well as platelet large cell count and ratio, than the control group. These findings concur with other studies that postulate that high parasitemias are known to exacerbate anaemia due to excessive haemolysis of parasitized RBCs [18, 19, 20, 21].

Furthermore, thrombocytopenia was also observed among the cases which has also been noted by previous studies that increasing levels of P. falciparum parasite density results in a decreased platelet count via peripheral destruction [22]. Another study suggested immune-mediated destruction of circulating platelets as a possible cause of thrombocytopenia in malaria infections, especially those caused by P. falciparum [23]. Another observation in this study was that the cases had a significantly higher neutrophil count than the control group. These findings parallel what was observed in the study of Kotepui et al., (2015) and is also consistent with the integral role that neutrophils play in the immune response to pathogens [24]. On another hand, the study recorded lower levels of eosinophils and basophils for the cases than the control group in line with other studies [24].

A key objective of this study was to determine whether significant differences exist between the control group and the cases with regards to their levels of CD4⁺, CD3⁺, CD8⁺, and CD45⁺ lymphoid cells. This was to understand how malaria parasites affect the normal profile of immune cells in the peripheral blood [25]. From the study outcome, it was seen that more participants in the control group had normal CD3⁺ cells as compared with the cases who showed depletion in the cells. A similar observation was made for CD8⁺, CD4⁺, CD45 + cell levels. This finding was in line with earlier reports that T cell lymphopenia is a well-established feature of P. falciparum malaria with the depletion attributed to sequestration or apoptosis [26, 27, 28, 29].

In determining the possible associations between the degree of malaria infection (parasite density) and specific leucocytic and haematological parameters among the cases, it was seen that with the exception of haematocrit levels, none of the parameters evaluated showed a significant association with parasite density. This is in line with the study of Bashawri et al., (2002) and Ekval, (2003), but in contrast to a previous study by McKenzie et al., (2005) which found a consistent positive relationship between leukocyte counts and parasite density in Plasmodium-infected patients [18, 19, 30].

The main conclusions of the study are that there was high parasite density among the cases suggestive of high intensity of infection among them. It was also observed that Plasmodium falciparum malaria infection is characterized by considerable haematological alterations in lymphocyte sub-sets (CD3, CD4, and CD8) tested in the current study in particular and it appears to be associated with CD4 + lymphocytopenia and also significantly associated with decreasing haematocrit levels.

The aim, design and setting of the study.

The aim of the study was to determine immunophenotypic alterations in the level of peripheral blood lymphocytes and their subsets in adults with uncomplicated P. falciparum malaria infection and apparently healthy participants.

This was a cross-sectional comparative study comprising of 414 participants (214 cases and 200 controls) conducted from April 2018 to June 2019 in two municipalities of the Volta region of Ghana, namely: Ketu South Municipal District and Ave Dakpa, the capital of the Akatsi North District.

The characteristics of participants.

The study comprised of malaria patients of 18 years and above who consented to take part in the study. Apparently healthy blood donors living in the study area were included as controls.

Sampling Technique

A convenient sampling method was used to recruit the participants where they were selected based on their convenient accessibility. Five (5) milliliters of venous blood were collected by venipuncture from each participant into an EDTA tube. Whole blood samples and slide smears were sent for analyses daily at the Ketu South Municipal Hospital laboratory and the Fevers Unit Laboratory of the Korle-Bu Teaching Hospital. Clinical and demographic data were also recorded using the structured questionnaire.

Laboratory Investigations

Laboratory procedures employed included serological, microscopy and haematological analysis.

Serology

Malaria Rapid Diagnostic Test kit was used for initial screening of participants before collection of venous blood samples. The manufacturer’s procedure was followed to carry out the test. Briefly, the cassette was labeled with patient identification (ID) number. The middle or ring finger of the patient was disinfected with 70% alcohol and allowed to air dry. The disinfected finger was pricked, and the first blood was wiped off with dry cotton. A capillary tube was held vertically to draw whole blood specimen. The blood was transferred into the sample well marked (S) on the cassette and buffer applied. Results were read within the time duration specified by the manufacturer. Following confirmation of positive malaria test, 5 ml of blood was taken into EDTA tube and used for the other tests.

Microscopy

Thick and thin blood films stained with 3% Giemsa stain were examined microscopically. Briefly, for thick film, a drop of blood (about 6µl in size) was placed on the center of a pre-labeled grease-free glass slide. Without delay, the blood was spread with a glass spreader held at a steep angle to achieve a thick smear in a circle the size of a dime (diameter 1-2cm). This was allowed to air dry in a horizontal position. Then Giemsa stain (3% solution diluted with buffer) was applied on the thick film and allowed to stand for 30 minutes. After this time, the stain was washed off using phosphate buffer (pH = 7.3–7.4) and again allowed to air dry. The dried stained thick smear was finally viewed under a light microscope using the 100x oil immersion objective lens.

For the thin films, 2µl of blood was placed on the Centre of a grease-free glass slide. The drop of blood was then spread with a glass spreader held at an angle of 45^o to obtain a thin film with a smooth tail end. This was air dried in a horizontal position and then fixed with absolute methanol for two minutes. After the fixing, Leishman/Giemsa stain was applied on the thin film and allowed to stain for 30 minutes. The stain was then washed off using phosphate buffer (pH = 7.2–7.4) and also air dried. The stained thin film was then viewed under a light microscope (Olympus Optics Ltd. UK) using 100x oil immersion objective lens for species identification.

Determination of Parasitaemia.

The prepared thick film was used for estimation of parasitaemia. The number of parasites/µl of blood was determined by enumerating the number of parasites in relation to total WBC count using the formula below:

Full Blood Count

For each blood sample taken, full blood count was done using full automated Haematology Analyzer following manufacturer’s instructions (Mindrey BC-5150 Auto Haematology Analyzer). The blood samples were placed under the aspiration chamber of the Haematology Analyzer. The aspiration button was pressed for them to suck a portion of the sample into the analyzer for the parameters to be measured. Other parameter results were obtained via calculations.

Flow Cytometric analysis

To obtain absolute counts of lymphocytes, a dualplatform method (using a haematology instrument and a flow cytometer) was applied. A BD FACScalibur flow cytometer (Becton Dickinson, NJ, USA) was used for identification and enumeration of lymphocyte subsets. Before data acquisition, instrument parameters were checked and optimized using CaliBRITE beads (Becton Dickinson, NJ, USA). Data was acquired with BD CellQuest Pro software (Becton Dickinson) and analyzed with Paint-A-Gate software followed by MultiSET (both from Becton Dickinson, NJ, USA). As a control for appropriate lymphocyte gating, the mean percentages of CD4⁺ and CD8⁺ T cells were checked to ensure that they fall within a 10% range of the average percentage of CD3⁺ T-cells.

Data Analysis

Data collected were entered into Microsoft Excel 2010 (Microsoft Corp., USA), and imported into STATA statistical software version 12 (STATA Corp, TX, USA) for analysis. Descriptive statistics were used in summarizing the data obtained. The significance of the differences between the malaria-infected and control participants regarding the proportions of their CD3+, CD4+, CD8+, and CD45 + levels were assessed by calculating the Z ratio. Furthermore, an independent-samples t-test was conducted to compare the two study groups for differences in the means of their haematological parameters. The eta squared statistic was computed to determine the effect size and interpreted based on the guidelines of Cohen (1988) that 0.01 represents a small effect, 0.06 represents a moderate effect, and 0.14 represents a large effect.

With variables whose data violated the assumption of equal variances (which needed to be satisfied when using the independent sample t-test), values of the test statistic that compensate for the violation of this assumption were computed. Finally, the Pearson product moment correlation was used to determine associations between parasite density and each of white blood cells count, platelet count, red blood cell count, haemoglobin and haematocrit levels. These associations were presented using the Pearson product moment correlation coefficient. P-values that were below 0.05 were considered statistically significant.

CD	Cluster of differentiation
CI	Confidence Interval
EDTA	Ethylenediaminetetraacetic
FBC	Full Blood Count
IFN	Interferon
ITNs	Insecticide Treated nets
P. falciparum	Plasmodium falciparum
SD	Standard deviation
TNF	Tissue Necrosis Factor
WHO	World Health Organization

Ethics approval and consent to participate.

Ethical clearance was obtained from the ethical and protocol review committee of the College of Health Sciences (CHS-Et/M.1-P5.3/2018-2019) as well as the institutional ethical committees of the Ketu South Municipal and the Korle-Bu Teaching Hospitals. All the study details were explained to the participants and informed consents were obtained before the commencement of the study. All methods were carried out in accordance with relevant guidelines and regulations.

Consent for publication

Not applicable

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

The study was funded using the University of Ghana book and research allowances of the research team members. The University of Ghana did not play any role as far as the design of the study, collection, analysis, and interpretation of data as well as writing of the manuscript are concerned.

Author’s contributions

SAB participated in the design, co-supervised the research, and drafted the manuscript. BTM participated in the design and co-supervised the work and proofread the manuscript. SAA participated in the design of the study and carried out the experimental work. DNOA carried out the data analysis and editing of the manuscript. SA-M/LA participated in the supervision of the work and proof reading of the manuscript. All authors read and approved the final manuscript.

Acknowledgements

We are grateful to the directors and laboratory managers of the Ketu South Municipal Hospital laboratory and the Fevers Unit Laboratory of the Korle-Bu Teaching Hospital for their assistance in carrying out this study.

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No competing interests reported.

Evaluation of Immunophenotypic Alterations of Peripheral Blood Lymphocytes and their Sub-Sets in Uncomplicated P. Falciparum Infection

Status:

Version 1

Abstract

Figures

Background

Results

Demographic and social features of the study participants

General Haematological Parameters Among The Study Participants

Associations Between Parasite Density And Haematological Parameters

Discussion

Conclusions

Methods

Sampling Technique

Laboratory Investigations

Serology

Microscopy

Full Blood Count

Data Analysis

Abbreviations

Declarations

References

Additional Declarations

Status:

Version 1