Red blood cell tension protects against severe malaria in the Dantu blood group.

Malaria has had a major effect on the human genome, with many protective polymorphisms-such as the sickle-cell trait-having been selected to high frequencies in malaria-endemic regions1,2. The blood group variant Dantu provides 74% protection against all forms of severe malaria in homozygous individuals3-5, a similar degree of protection to that afforded by the sickle-cell trait and considerably greater than that offered by the best malaria vaccine. Until now, however, the protective mechanism has been unknown. Here we demonstrate the effect of Dantu on the ability of the merozoite form of the malaria parasite Plasmodium falciparum to invade red blood cells (RBCs). We find that Dantu is associated with extensive changes to the repertoire of proteins found on the RBC surface, but, unexpectedly, inhibition of invasion does not correlate with specific RBC-parasite receptor-ligand interactions. By following invasion using video microscopy, we find a strong link between RBC tension and merozoite invasion, and identify a tension threshold above which invasion rarely occurs, even in non-Dantu RBCs. Dantu RBCs have higher average tension than non-Dantu RBCs, meaning that a greater proportion resist invasion. These findings provide both an explanation for the protective effect of Dantu, and fresh insight into why the efficiency of P. falciparum invasion might vary across the heterogenous populations of RBCs found both within and between individuals.

Dantu Blood Group Erythrocytes Form Large Plasmodium falciparum Rosettes Less Commonly.

Dantu erythrocytes, which express a hybrid glycophorin B/A protein, are protective against severe malaria. Recent studies have shown that Dantu impairs Plasmodium falciparum invasion by increasing erythrocyte membrane tension, but its effects on pathological host-parasite adhesion interactions such as rosetting, the binding of uninfected erythrocytes to P. falciparum-infected erythrocytes, have not been investigated previously. The expression of several putative host rosetting receptors-including glycophorin A (GYPA), glycophorin C (GYPC), complement receptor 1 (CR1), and band 3, which complexes with GYPA to form the Wrightb blood group antigen-are altered on Dantu erythrocytes. Here, we compare receptor expression, and rosetting at both 1 hour and 48 hours after mixing with mature trophozoite-stage Kenyan laboratory-adapted P. falciparum strain 11019 parasites in Dantu and non-Dantu erythrocytes. Dantu erythrocytes showed lower staining for GYPA and CR1, and greater staining for band 3, as observed previously, whereas Wrightb and GYPC staining did not vary significantly. No significant between-genotype differences in rosetting were seen after 1 hour, but the percentage of large rosettes was significantly less in both Dantu heterozygous (mean, 16.4%; standard error of the mean [SEM], 3.2) and homozygous donors (mean, 15.4%; SEM, 1.4) compared with non-Dantu erythrocytes (mean, 32.9%; SEM, 7.1; one-way analysis of variance, P = 0.025) after 48 hours. We also found positive correlations between erythrocyte mean corpuscular volume (MCV), the percentage of large rosettes (Spearman's rs = 0.5970, P = 0.0043), and mean rosette size (rs = 0.5206, P = 0.0155). Impaired rosetting resulting from altered erythrocyte membrane receptor expression and reduced MCV might add to the protective effect of Dantu against severe malaria.

The Dantu blood group prevents parasite growth in vivo: Evidence from a controlled human malaria infection study.

Background: The long co-evolution of Homo sapiens and Plasmodium falciparum has resulted in the selection of numerous human genetic variants that confer an advantage against severe malaria and death. One such variant is the Dantu blood group antigen, which is associated with 74% protection against severe and complicated P. falciparum malaria infections in homozygous individuals, similar to that provided by the sickle haemoglobin allele (HbS). Recent in vitro studies suggest that Dantu exerts this protection by increasing the surface tension of red blood cells, thereby impeding the ability of P. falciparum merozoites to invade them and reducing parasite multiplication. However, no studies have yet explored this hypothesis in vivo. Methods: We investigated the effect of Dantu on early phase P. falciparum (Pf) infections in a controlled human malaria infection (CHMI) study. 141 sickle-negative Kenyan adults were inoculated with 3.2 × 103 aseptic, purified, cryopreserved Pf sporozoites (PfSPZ Challenge) then monitored for blood-stage parasitaemia for 21 days by quantitative polymerase chain reaction (qPCR)analysis of the 18S ribosomal RNA P. falciparum gene. The primary endpoint was blood-stage P. falciparum parasitaemia of ≥500/µl while the secondary endpoint was the receipt of antimalarial treatment in the presence of parasitaemia of any density. On study completion, all participants were genotyped both for Dantu and for four other polymorphisms that are associated with protection against severe falciparum malaria: α+-thalassaemia, blood group O, G6PD deficiency, and the rs4951074 allele in the red cell calcium transporter ATP2B4. Results: The primary endpoint was reached in 25/111 (22.5%) non-Dantu subjects in comparison to 0/27 (0%) Dantu heterozygotes and 0/3 (0.0%) Dantu homozygotes (p=0.01). Similarly, 49/111 (44.1%) non-Dantu subjects reached the secondary endpoint in comparison to only 7/27 (25.9%) and 0/3 (0.0%) Dantu heterozygotes and homozygotes, respectively (p=0.021). No significant impacts on either outcome were seen for any of the other genetic variants under study. Conclusions: This study reveals, for the first time, that the Dantu blood group is associated with high-level protection against early, non-clinical, P. falciparum malaria infections in vivo. Learning more about the mechanisms involved could potentially lead to new approaches to the prevention or treatment of the disease. Our study illustrates the power of CHMI with PfSPZ Challenge for directly testing the protective impact of genotypes previously identified using other methods. Funding: The Kenya CHMI study was supported by an award from Wellcome (grant number 107499). SK was supported by a Training Fellowship (216444/Z/19/Z), TNW by a Senior Research Fellowship (202800/Z/16/Z), JCR by an Investigator Award (220266/Z/20/Z), and core support to the KEMRI-Wellcome Trust Research Programme in Kilifi, Kenya (203077), all from Wellcome. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. For the purpose of Open Access, the authors have applied a CC BY public copyright license to any Author Accepted Manuscript version arising from this submission. Clinical trial number: NCT02739763.

Pooled testing conserves SARS-CoV-2 laboratory resources and improves test turn-around time: experience on the Kenyan Coast.

Background. International recommendations for the control of the coronavirus disease 2019 (COVID-19) pandemic emphasize the central role of laboratory testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent, at scale. The availability of testing reagents, laboratory equipment and qualified staff are important bottlenecks to achieving this. Elsewhere, pooled testing (i.e. combining multiple samples in the same reaction) has been suggested to increase testing capacities in the pandemic period. Methods. We discuss our experience with SARS-CoV-2 pooled testing using real-time reverse transcription polymerase chain reaction (RT-PCR) on the Kenyan Coast. Results. In mid-May, 2020, our RT-PCR testing capacity for SARS-CoV-2 was improved by ~100% as a result of adoption of a six-sample pooled testing strategy. This was accompanied with a concomitant saving of ~50% of SARS-CoV-2 laboratory test kits at both the RNA extraction and RT-PCR stages. However, pooled testing came with a slight decline of test sensitivity. The RT-PCR cycle threshold value (ΔCt) was ~1.59 higher for samples tested in pools compared to samples tested singly. Conclusions. Pooled testing is a useful strategy to increase SARS-CoV-2 laboratory testing capacity especially in low-income settings.