Evaluation of splenic accumulation and colocalization of immature reticulocytes and Plasmodium vivax in asymptomatic malaria: A prospective human splenectomy study

Abstract

Background A very large biomass of intact asexual-stage malaria parasites accumulates in the spleen of asymptomatic human individuals infected with Plasmodium vivax. The mechanisms underlying this intense tropism are not clear. We hypothesised that immature reticulocytes, in which P. vivax develops, may display high densities in the spleen, thereby providing a niche for parasite survival. Methods and findings We examined spleen tissue in 22 mostly untreated individuals naturally exposed to P. vivax and Plasmodium falciparum undergoing splenectomy for any clinical indication in malaria-endemic Papua, Indonesia (2015 to 2017). Infection, parasite and immature reticulocyte density, and splenic distribution were analysed by optical microscopy, flow cytometry, and molecular assays. Nine non-endemic control spleens from individuals undergoing spleno-pancreatectomy in France (2017 to 2020) were also examined for reticulocyte densities. There were no exclusion criteria or sample size considerations in both patient cohorts for this demanding approach. In Indonesia, 95.5% (21/22) of splenectomy patients had asymptomatic splenic Plasmodium infection (7 P. vivax, 13 P. falciparum, and 1 mixed infection). Significant splenic accumulation of immature CD71 intermediate- and high-expressing reticulocytes was seen, with concentrations 11 times greater than in peripheral blood. Accordingly, in France, reticulocyte concentrations in the splenic effluent were higher than in peripheral blood. Greater rigidity of reticulocytes in splenic than in peripheral blood, and their higher densities in splenic cords both suggest a mechanical retention process. Asexual-stage P. vivax-infected erythrocytes of all developmental stages accumulated in the spleen, with non-phagocytosed parasite densities 3,590 times (IQR: 2,600 to 4,130) higher than in circulating blood, and median total splenic parasite loads 81 (IQR: 14 to 205) times greater, accounting for 98.7% (IQR: 95.1% to 98.9%) of the estimated total-body P. vivax biomass. More reticulocytes were in contact with sinus lumen endothelial cells in P. vivax- than in P. falciparum-infected spleens. Histological analyses revealed 96% of P. vivax rings/trophozoites and 46% of schizonts colocalised with 92% of immature reticulocytes in the cords and sinus lumens of the red pulp. Larger splenic cohort studies and similar investigations in untreated symptomatic malaria are warranted. Conclusions Immature CD71+ reticulocytes and splenic P. vivax-infected erythrocytes of all asexual stages accumulate in the same splenic compartments, suggesting the existence of a cryptic endosplenic lifecycle in chronic P. vivax infection. Findings provide insight into P. vivax-specific adaptions that have evolved to maximise survival and replication in the spleen.

Positron emission tomography and magnetic resonance imaging in experimental human malaria to identify organ-specific changes in morphology and glucose metabolism: A prospective cohort study

“…In a nonhuman primate P. vivax model, spleendependent expression of molecules mediating adhesion to human splenic fibroblasts has been described [36], suggesting the presence of a parasite phenotype that may favor splenic accumulation. Finally, very recent data suggest that immature reticulocytes accumulate in the human spleen providing a favorable niche for P. vivax accumulation [11]. We now show evidence supporting splenic tropism and splenic accumulation of P. vivax very early in infection.…”

“…In these studies, few or no parasites were identified in the brain or intestines, whereas the bone marrow and liver sinusoids were major tissue reservoirs for P. vivax schizonts and gametocytes [9]. An apparent predilection for the spleen, bone marrow, and liver is consistent with biomarker evidence of a hidden biomass of parasite accumulating in non-endothelial lined spaces [10] and the strict reticulocyte tropism of this species, which may promote concentration in organs in which immature reticulocytes accumulate [4,11]. Nonetheless, this hypothesis is challenging to confirm in life, where these processes are largely inaccessible to evaluation.…”

Lessons Learned for Pathogenesis, Immunology, and Disease of Erythrocytic Parasites: Plasmodium and Babesia

“…Recently, Kho et al. described the accumulation of biomass due to medium to high CD71 + expressing reticulocytes as well as non-phagocytosed parasites, thus providing another shelter for the parasite’s survival and replication, contributing to splenomegaly ( Kho et al., 2021 ). We also demonstrated the presence of free parasites in the spleens during the murine model of B. microti infection ( Djokic et al., 2018a ); however, neither RBC aggregations nor biomass accumulation was observed ( Srivastava et al., 2015 ).…”

Zoonotic Malaria: Non-Laverania Plasmodium Biology and Invasion Mechanisms

“…Splenic tropism is the highest in P. vivax infections with more than 98% of asexual-stage parasites. This may suggest that chronic malaria could be due to the infection of the spleen, instead of blood infection solely [ 90 , 91 ]. Infected erythrocytes deformability can also be observed in P. ovale and P. cynomolgi infections [ 87 ].…”