Demographic and Laboratory Characteristics of all Study participants
Children (n = 217, aged 1-192 months) with acute febrile conditions (temperature > 37.5 °C) were enrolled in the study. The participants were first broadly categorized based on malaria infection status upon microscopic examination and demonstration of any asexual form of P. falciparum malaria (trophozoite or schizont) as non-infected (n = 132) and infected (n = 85). Sex (P = 0.240) and age (P = 0.143) were comparable between the two groups.
Distribution of HBB rs334 genotypes in the clinical groups was also determined. Among the malaria negative patients, 90 (68.2%) had HbAA, 31 (23.5%) had HbAS and 11 (8.3%) had HbSS genotypes while in the infected group, 58 (68.2%) has HbAA, 14 (16.5%) had HbAS and 13 (15.3%) had HbSS genotype. Generally, haemoglobin types were comparable with regard to P. falciparum infection, P = 0.743. The overall genotype distribution for the HBB rs334 deviated significantly from the HWE (χ2 = 32.02, P < 0.001) with a minor allele frequency of 0.21.
Heamatological markers for anaemia i.e. haemoglobin, haematocrit and RBC count in those without P. falciparum parasitaemia were higher than those infected with P. falciparum malaria [(median (IQR), 10.5 (2.3) and 10.1 (3.2), P = 0.022], [(median (IQR), 35.2 (6.8) and 32.8 (9.0), P = 0.009] and [(median (IQR), 4.8 (0.7) and 4.6 (1.3), P = 0.045], respectively. Red cell distribution width (RDW, P = 0.703), Mean Corpuscular Volume (MCV, P = 0.349), Mean Corpuscular Heamoglobin (MCH, P = 0.744), Mean Corpuscular Haemoglobin Concentration (MCHC, P = 0.479), white blood cells (WBC, P = 0.746), lymphocytes (P = 0.103), monocytes (P = 0.084), and granulocytes (P = 0.354) counts were comparable between the two groups (Table 1). Further analysis revealed that the platelet counts were lower in the infected group [median (IQR), 236 (129.5) vs. non-infected group [median (IQR), 278 (112.8), P = 0.001, respectively. The mean platelet volume (MPV) were however, not different between the groups (P = 0.119). The children with malaria infection further had reduced plateletcrit (PCT), infected [median (IQR), 0.13 (0.1) vs. non-infected [median (IQR), 0.2 (0.1), P = 0.001. The platelet distribution width (PDW) were, however, comparable between the groups (P = 0.629).
Table 1
General Demographic and Laboratory Characteristics of the Study Participants
All the study participants (Infected and non-infected) | P. falciparum infected children based on genotype (n = 85) |
Characteristics | Non-Infected (n = 132) | Infected (n = 85) | P-value | HbAA (n = 58) | HBAS (n = 14) | HbSS (n = 13) | P-Value |
Sex, n (%) | | | | | | | |
Male Female | 76 (57.6) | 42 (49.4) | 0.240a | 30 (51.7) | 7 (50) | 8 (61.5) | 0.690a |
56 (42.4) | 43 (50.6) | 28 (48.3) | 7 (50) | 5 (38.5) | |
Age, (months) | 30 (48) | 36 (55) | 0.143b | 36 (64.5) | 36 (32.3) | 16 (27) | 0.660c |
Haemoglobin type | | | | |
HbAA, n (%) | 90 (68.2) | 58 (68.2) | | | | | |
HbAS, n (%) | 31 (23.5) | 14 (16.5) | 0.743a | | | | |
HbSS, n (%) | 11 (8.3) | 13 (15.3) | | | | | |
Haematological Parameters | Haematological Parameters |
Haemoglobin, gdL− 1 | 10.5 (2.3) | 10.1 (3.1) | 0.022b | 10.2 (2.2) | 10.4 (1.6) | 7.3 (1.3) | 0.001c |
Haematocrit, % | 35.2 (6.8) | 32.8 (9.0) | 0.009b | 34.6 (8.3) | 32.8 (6.0) | 26.4 (4.4) | 0.009c |
RBC, (× 1012µL− 1) | 4.8 (0.7) | 4.6 (1.3) | 0.045b | 4.6 (1.0) | 4.8 (1.5) | 3.2 (1.7) | 0.048c |
RDW, % | 11.7 (3.0) | 11.6 (2.8) | 0.703b | 11.3 (2.2) | 11.5 (2.6) | 14.9 (3.3) | 0.030c |
MCV, fL | 76.3 (11.4) | 75.2 (10.9) | 0.349b | 75.2 (9.9) | 74.1 (11.8) | 79.5 (15.9) | 0.415c |
MCH, fL/cell | 22.7 (4.3) | 22.5 (3.7) | 0.744b | 22.1 (3.7) | 22.6 (4.0) | 23.1 (3.5) | 0.724c |
MCHC, gdL− 1 | 29.6 (3.7) | 29.8 (3.0) | 0.479b | 29.7 (2.5) | 30.9 (1.7) | 27.7 (4.1) | 0.054c |
WBC (×103µL− 1) | 8.5 (6.0) | 8.6 (5.2) | 0.746b | 7.8 (4.8) | 9.58 (5.2) | 12.68 (4.6) | 0.078c |
Lymphocytes, (×103µL− 1) | 44.1 (25.3) | 38 (66.9) | 0.103b | 36.55 (27.2) | 37 (36.2) | 50.9 (29.1) | 0.596c |
Monocytes, (×103µL− 1) | 9.3 (4.4) | 11.2 (7.2) | 0.084b | 11.2 (7.4) | 12 (7.8) | 8.1 (6.1) | 0.282c |
Granulocytes, (×103µL− 1) | 46.1 (26.3) | 50.0 (25.8) | 0.354b | 51.4 (27.2) | 48.9 (27.5) | 39 (28.2) | 0.494c |
Platelet Counts, (×103µL− 1) | 278.0 (112.8) | 236 (129.5) | 0.001b | 220 (1127) | 233 (129) | 236(140) | 0.399c |
MPV, fL | 5.5 (0.5) | 5.40 (0.6) | 0.119b | 5.4 (0.6) | 5.3 (0.9) | 5.2 (0.8) | 0.990c |
PCT, % | 0.2 (0.1) | 0.13 (0.1) | 0.001b | 0.13 (0.1) | 0.14 (0.1) | 0.12 (0.1) | 0.772c |
PDW, % | 9.4 (1.5) | 9.5 (1.5) | 0.629b | 9.6 (1.4) | 9.5 (2.0) | 9.5 (0.40) | 0.951c |
Data are presented as the median (interquartile range; IQR) values unless stated otherwise. Study participants were categorized into non-infected and infected (with any density parasitaemia). a Statistical significance was determined by the Chi-square (χ2) analysis. b Statistical significance was determined using Mann- Whitney test. Abbreviations: HbAA; Normal haemoglobin; HbAS: Heterozygous sickle cell trait; HbSS: Homozygous Haemoglobin S; MCV: Mean corpuscular volume: MCH; mean corpuscular haemoglobin: MCHC; mean corpuscular haemoglobin concentration: WBC; White blood cell count: RBC; Red blood cells: RDW; Red cell distribution width: MPV; mean platelet volume: PCT; Plateletcrit: PDW: Platelet distribution width. |
Haematocrit levels were significantly higher in HbAA [median (IQR), 34.6 (8.3)] and HbAS [median (IQR), 32.8 (6.0)] relative to HbSS [median (IQR), 26.4 (4.4), P = 0.009]. Furthermore, red blood cell (RBC) count were equally significantly higher in HbAA [median (IQR), 4.6 (1.0)] and HbAS [median (IQR), 4.8 (1.5)] compared to HbSS [median (IQR), 3.2(1.7), P = 0.048]. Nonetheless, Red Cell Distribution Width (RDW) was relatively lower in HbAA [median (IQR), 11.3 (2.2)] and HbAS [median (IQR), 11.5 (2.6)] compared to HbSS [median (IQR), 14.9 (3.3), P = 0.030]. In cases where significant differences were detected using the Kruskal-Wallis test, we performed a post-hoc pairwise analysis using Dunn’s multiple comparison test. The post-hoc test was run on haemoglobin, haematocrit red blood cell count and red cell distribution width. Our results revealed that haemoglobin was reduced in children with HbSS than those with HbAS (P = 0.001). Furthermore, haematocrit was markedly reduced in children with HbSS than those with HbAS (P = 0.002). Red blood cell count was reduced in children with HbSS than those with HbAS (P = 0.023). However, red cell distribution width was highly raised in children with HbSS than those with HbAS (P = 0.001). There were no significant differences in the levels of other hematological indices and carriage of different HBB genotypes in children with P. falciparum malaria. These results are summarized in Table 1.
Haematological abnormalities in carriage of different sickle cell genotypes in P. falciparum infection
Haematological abnormalities in malaria infection and in sickle cell disease are as a result of benign changes in haematological parameters [22]. Major haematological abnormalities that have been found to be associated with malaria include anaemia, thrombocytopenia, leucocytosis and monocytosis [22]. To determine whether carriage of different sickle cell genotypes have effect on the severity of haematological abnormalities once an individual is infected, we evaluated their severity in children with P. falciparum infection and carriage of different sickle cell genotypes. Anaemia was defined according to the World Health Organisation’s criteria as a condition where haemoglobin level < 11 g/dl in children [23]. These severities were considered as described herein; based on haemoglobin level alone, severity of anaemia was categorised into groups of severe (haemoglobin level < 7 g/dl) mild (haemoglobin level 10-10.9 g/dl), moderate (haemoglobin level 7-9.9 g/dl) and non-anaemic (haemoglobin level > 11 g/dl) [13]. Severe anaemia was seen in 8.6% of children with HbAA, 7.1% with HbAS and 23.1% of those with HbAA. Moderate anaemia was seen in 34.5%, 21.4% and 69.2% of HbAA, HbAS, and HbSS, respectively. Mild anaemia was detected in 22.4% in HbAA, 35.7% in HbAS and none in HbSS. Otherwise, non-anaemic children were also seen in 34.5%, 37.5% and 7.7% in those with HbAA, HbAS and HbSS genotypes, respectively.
Thrombocytopenia was defined as a haematological condition in which platelet count is below 150×103 µL− 1 of blood [24]. For evaluation purposes, severity of thrombocytopenia was categorized as severe (platelet count < 50×103 µL− 1), moderate (platelet count between 50–100×103 µL− 1) and mild (platelet count between 100–150×103 µL− 1). Otherwise, any platelet count above 150×103 µL− 1 was considered non-thrombocytopenic. None of the participants demonstrated severe thrombocytopenia in any of the HBB genotypes. Moderate thrombocytopenia was seen in 5.2% and 7.1% of HbAA and HbAS, respectively. However, none had thrombocytopenia in children with HbSS genotype. Mild thrombocytopenia was demonstrated in 15.5%, 7.1% and 15.4% in individuals with HbAA, HbAS and HbSS, respectively. However, non-thrombocytopenia was demonstrated in 79.3% of children with HbAA, 85.7% of children with HbAS and 11% of children with HbSS.
Leucocytopenia was defined as a condition in which total leucocyte count is below 4×103 µL− 1 of blood. There was no leucocytopenia in any of the study participants. Leucocytosis was defined as a benign abnormality in which total leucocyte count is above 11×103 µL− 1) of blood [24]. Leucocytosis was demonstrated in 31% of children with HbAA, 42.9% in children with HbAS and in 69.2% of children with HbSS. Normal leucocyte count (WBC count between (4–11×103 µL− 1) was seen in 70% of those with HbAA genotype, 57.1% in those with HbAS and in 30.8% in those with HbSS genotype. Monocytosis was defined as a condition in which total monocyte count is below 12% of the total white blood cell count in peripheral blood [24, 25]. Monocytosis was seen in 59% in children with HbAA, 79% in children with HbAS and in 31% in children with HbSS genotype. The distribution of severity of anaemia, thrombocytopenia, leucocytosis, leucocytopaenia and monocytosis are summarized in Table 2.
Table 2
Distribution and severity of selected haematological abnormalities with regard to haemoglobin type in P. falciparum infected children
Haematological parameter | Severity of the | HBB genotypes | Total n (%) |
HbAA n (%) | HbAS n (%) | HbSS n (%) |
Haemoglobin | Severe anaemia | 5 (8.6) | 1 (7.1) | 3 (23.1) | 9 (10.5) |
Moderate anaemia | 20 (34.5) | 3 (21.4) | 9 (69.2) | 32 (42.4) |
Mild anaemia | 13 (22.4) | 5 (35.7) | 0 (0) | 18 (21.2) |
Non-anaemic | 20 (34.5) | 5 (35.7) | 1 (7.7) | 25 (29.4) |
| Total | 58 | 14 | 13 | 85 |
Platelets | Severe thrombocytopenia | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
Moderate thrombocytopenia | 3 (5.2) | 1 (7.1) | 0 (0) | 4 (4.7) |
Mild thrombocytopenia | 9 (15.5) | 1 (7.1) | 2 (15.4) | 12 (14.1) |
| Non-thrombocytopenia | 46 (79.3) | 12 (85.7) | 11 (84.6) | 69 (81.2) |
| Total | 58 | 14 | 13 | 85 |
Leucocytes | Leucocytopaenia | 0 (0) | 0 (0) | 0 (0) | 0 (0) |
Leucocytosis | 18 (31) | 6 (42.9) | 9 (69.2) | 33 (38.8) |
Normal | 40 (70) | 8 (57.1) | 4 (30.8) | 52 (61.2) |
| Total | 58 | 14 | 13 | 85 |
Monocytes | Monocytosis | 34 (59) | 11(79) | 4 (31) | 49 (58) |
| Normal | 24 (41) | 3 (21) | 9 (69) | 36 (42) |
| | 58 | 14 | 13 | 85 |
Data are presented as proportions (n) and percentages (%) of different HBB genotypes and different severities of haematological abnormalitis in children infected with P.falciparum malaria. |
Correlation analysis
Mutations in haemoglobin beta sub-unit gene (HBB) have been found to primarily affect the red blood cells structurally resulting into sickle cell and physiologically affecting normal red cell functions [22]. However, it is not known how primary red cell measurement correlate with red cell indices in individuals infected with P. falciparum and presenting with different HBB genotypes. We used a partial correlation test while controlling for age and sex, factors previously shown to affect haematological indices in a population [26].
In the HbSS genotype group, the RBC count revealed a negative correlation with RDW in children malaria-infected and non-infected (r= -0.750, P = 0.008 and r= -0.634, P = 0.049, respectively). Furthermore, there was a negative correlation between RBC count and both MCV and MCH in malaria-infected children (r= -0.833, P = 0.001) and (r= -0.750, P = 0.005), respectively. Haematocrit was negatively correlated with RDW in both malaria-infected and non-infected children (r= -0.918, P = 0.001) and (r= -0.718, P = 0.009), respectively. Additional analysis in the carriage HbAS genotype, showed a negative correlation between haemoglobin with RDW in malaria uninfected children (r= -0.694, P < 0.0001). The RBC count showed a negative correlation with MCV (r= -0.752, P = 0.003) and with MCH (r= -0.797, P = 0.001) in malaria-infected children.
In HbAA genotype, RBC count showed a negative correlation with MCV (r= -0.324, P = 0.002), MCH (r= -0.273, P = 0.010), MCHC (r= -0.401, P = 0.000) and RDW (r= -0.235, P = 0.027) in children without malaria infection. Haematocrit showed a positive correlation with MCV (r = 0.524, P < 0.0001), and a negative correlation with MCHC (r= -0.333, P = 0.001) and RDW (r=-0.361, P < 0.0001) in children without malaria infection. Haemoglobin showed a positive correlation with MCV (r = 0.525, P < 0.0001), MCH (r = 0.408, P < 0.0001) and MCHC (r = 0.269, P = 0.011), however, it showed a negative correlation with RDW (r = -0.664, P < 0.0001) in children without malaria infection. The RBC showed a negative correlation with MCV (r= -0.393, P = 0.003), MCH (r= -0.408, P = 0.002), MCHC (r= -0.270, P = 0.043) and RDW (r= -0.399, P = 0.002) in malaria-infected children. There was a negative correlation between haematocrit and PDW (r= -0.446, P = 0.001) in children infected with malaria. Haemoglobin showed positive correlation with MCH (r = 0.317, P = 0.016) and MCHC (r = 0.269, P = 0.011) in children infected with P. falciparum malaria. Further analysis revealed a negative correlation between haemoglobin and RDW (r= -0.479, P = 0.001) in children infected with P. falciparum malaria and (r= -0.479, P = 0.001) in children without P. falciparum malaria. It is therefore probable that the RBC count, haematocrit and haemoglobin concentration decrease with increase in RDW levels in P. falciparum-infected children with carriage of HbSS genotype relative to other HBB genotypes. Correlation between primary red cell measurements and red cell indices are summarized in Table 3.
Table 3
Correlation of haematological parameters with Sickle cell genotype in children with and without P. falciparum infection
| Malaria negative (N = 132) | Malaria positive (N = 85) |
Control Variable | | MCV (fL) | MCH (Pg) | MCHC (gdL−) | RDW (%) | MCV (fL) | MCH (Pg) | MCHC (gdL−) | RDW (%) |
Sickle cell anaemia (HbSS) (n = 11) | Sickle cell anaemia (HbSS) (n = 13) |
Age and sex | RBC count | r | -0.362 | -0.068 | 0.060 | -0.634 | -0.883 | -0.750 | -0.118 | -0.725 |
P value | 0.304 | 0.852 | 0.870 | 0.049 | 0.001 | 0.005 | 0.715 | 0.008 |
Haematocrit | r | 0.231 | 0.172 | -0.086 | -0.918 | -0.555 | -0.418 | 0.273 | -0.718 |
P value | 0.521 | 0.635 | 0.813 | < 0.0001 | 0.061 | 0.176 | 0.391 | 0.009 |
Haemoglobin | r | -0.218 | 0.032 | 0.563 | -0.616 | -0.526 | -0.220 | 0.521 | -0.792 |
P value | 0.545 | 0.929 | 0.090 | 0.058 | 0.079 | 0.493 | 0.082 | 0.002 |
| Sickle cell trait (HbAS) (n = 31) | Sickle cell trait (HbAS) (n = 14) |
Age and sex | RBC count | r | -0.299 | -0.494 | -0.434 | -0.292 | -0.752 | -0.797 | -0.305 | -0.392 |
P value | 0.116 | 0.006 | 0.019 | 0.125 | 0.003 | 0.001 | 0.311 | 0.185 |
Haematocrit | r | 0.515 | 0.221 | -0.458 | -0.507 | -0.269 | -0.369 | -0.394 | -0.506 |
P value | 0.004 | 0.249 | 0.013 | 0.005 | 0.375 | 0.214 | 0.183 | 0.078 |
Haemoglobin | r | 0.547 | 0.533 | 0.069 | -0.694 | -0.247 | -0.235 | -0.080 | -0.475 |
P value | 0.002 | 0.003 | 0.720 | < 0.0001 | 0.417 | 0.441 | 0.796 | 0.101 |
| Normal haemoglobin (HbAA) (n = 90) | Normal haemoglobin (HbAA) (n = 58) |
Age and sex | RBC count | r | -0.324 | -0.273 | -0.401 | -0.235 | -0.393 | -0.408 | -0.270 | -0.399 |
P value | 0.002 | 0.010 | < 0.0001 | 0.027 | 0.003 | 0.002 | 0.043 | 0.002 |
Haematocrit | r | 0.524 | 0.143 | -0.333 | -0.361 | 0.139 | .076 | -0.072 | -0.446 |
P value | < 0.0001 | 0.181 | 0.001 | < 0.0001 | 0.302 | 0.573 | 0.593 | 0.001 |
Haemoglobin | r | 0.525 | 0.408 | 0.269 | -0.664 | 0.257 | 0.317 | 0.285 | -0.479 |
P value | < 0.0001 | < 0.0001 | 0.011 | < 0.0001 | 0.053 | 0.016 | 0.032 | 0.001 |
Data are the partial correlations (r). Malaria negative patients (n = 132) and malaria positive patients (n = 85) with acute febrile illness were categorized on the basis of haemoglobin type. All statistical significance was determined by the partial correlation test (r) controlling for age and sex. Values in bold are statistically significant at P ≤ 0.05. Abbreviations: MCV; Mean corpuscular volume; MCH; mean corpuscular haemoglobin: MCHC; mean corpuscular haemoglobin concentration; RBC-Red blood cells, RDW; Red cell distribution width. r: the measure of strength of Pearson’s correlation. |