Comparative analysis of asexual and sexual stage Plasmodium falciparum development in different red blood cell types

Abstract

Background: Red blood cell (RBC) polymorphisms are suggested to influence the course of Plasmodium falciparum malaria. Whereas some variants have been found to be protective, others have been found to enhance parasite development. This study evaluated the effect of variant haemoglobin (Hb) and ABO blood groups on P. falciparum merozoite invasion, multiplication rates as well as gametocyte development.

Methods: Approximately 2.5 mL of venous blood was collected from each participant. Flow cytometry was used to determine the in vitro merozoite invasion rates of NF54 parasites into the blood of 66 non-parasitaemic individuals with variant Hb genotypes (HbSS, HbSC) and blood groups (A, B, O), which were then compared with invasion into HbAA blood. The ex vivo asexual parasite multiplication and gametocyte production rates of parasites from 79 uncomplicated malaria patients with varying Hb genotypes (HbAS, HbAC and HbAA) were also estimated using microscopy.

Results: Merozoite invasion rates were significantly reduced by about 50% in RBCs containing HbSS and HbSC relative to HbAA cells. The presence of blood group O and B reduced the invasion rates of HbSS by about 50% and 60%, respectively, relative to HbSC but the presence of blood group A removed the inhibitory effect of HbSS. The initial parasite densities in uncomplicated malaria patients with Hb genotypes HbAS and HbAC cells were similar but significantly lower than those with genotype HbAA. The ex vivo parasite multiplication rate, gametocytaemia and gametocyte conversion rates followed a similar trend but did not reach statistical significance (p > 0.05).

Conclusions: Parasite invasion rate into erythrocytes is dependent on both erythrocyte blood group antigen and haemoglobin genotype as blood group O and B provided protection via reduced merozoite invasion in RBCs containing HbSS relative to HbSC. Regardless of haemoglobin type, greater than 70% malaria patients had circulating ring stage parasites that differentiated into stage II gametocytes in 4 days.

Keywords: Gametocyte; Haemoglobinopathies; Malaria; Merozoite.

Fig. 1

Ex vivo parasite development in normal and variant Hb erythrocytes. a Parasite density on D0, b asexual parasite multiplication rates, c gametocytaemia on D4 and d gametocyte conversion rate. a Giemsa-stained thin blood smears prepared from AS n = 15, AC n = 19 and AA n = 45 D0 samples were used to determined asexual parasitaemia. b The fold increase in asexual parasite densities in samples counted on D6 were compared to that counted in the same culture on D4 from AS n = 12, AC n = 14 and AA n = 40 samples. c Gametocyte densities in samples from AS n = 12, AC n = 14 and AA n = 43 examined on D4. d D4 Gametocyte conversion rate, represented by the % of the D0 ring stage parasites that develop into early stage (II) gametocytes by D4 from AS n = 12, AC n = 14 and AA n = 43 samples. Parasite infected RBCs in the thin blood smears were counted against 1000 RBCs for asexual parasite density estimation and 20,000 RBCs for gametocyte densities

Fig. 2

Parasite invasion into normal and variant Hb erythrocytes. a Invasion of NF54 into erythrocytes with variant (HbSS n = 35 and HbSC n = 31) and normal (HbAA n = 4) Hb genotypes. The assay was performed in duplicate. b Invasion rates in HbSS and HbSC samples with different blood groups, ‘O’ n = 38, ‘B’ n = 14 and ‘A’ n = 14. c Invasion in HbSS (‘O’ n = 24, ‘A’ n = 7, ‘B’ n = 4) and HbSC (‘O’ n = 14, ‘A’ n = 7, ‘B’ n = 10) RBCs categorized according to blood group antigen. The data in the graphs represent the median and the error bars are the interquartile range