Retention of uninfected red blood cells causing congestive splenomegaly is the major mechanism of anemia in malaria.

Splenomegaly frequently occurs in patients with Plasmodium falciparum (Pf) or P. vivax (Pv) malarial anemia, but mechanisms underlying this co-occurrence are unclear. In malaria-endemic Papua, Indonesia, we prospectively analyzed red blood cell (RBC) concentrations in the spleen and spleen-mimetic retention in 37 subjects splenectomized for trauma or hyperreactive splenomegaly, most of whom were infected with Plasmodium. Splenomegaly (median 357 g [range: 80-1918 g]) was correlated positively with the proportion of red-pulp on histological sections (median 88.1% [range: 74%-99.4%]; r = .59, p = .0003) and correlated negatively with the proportion of white-pulp (median 8.3% [range: 0.4%-22.9%]; r = -.50, p = .002). The number of RBC per microscopic field (>95% uninfected) was correlated positively with spleen weight in both Pf-infected (r = .73; p = .017) and Pv-infected spleens (r = .94; p = .006). The median estimated proportion of total-body RBCs retained in Pf-infected spleens was 8.2% (range: 1.0%-33.6%), significantly higher than in Pv-infected (2.6% [range: 0.6%-23.8%]; p = .015) and PCR-negative subjects (2.5% [range: 1.0%-3.3%]; p = .006). Retained RBCs accounted for over half of circulating RBC loss seen in Pf infections. The proportion of total-body RBC retained in Pf- and Pv-infected spleens correlated negatively with hemoglobin concentrations (r = -.56, p = .0003), hematocrit (r = -.58, p = .0002), and circulating RBC counts (r = -.56, p = .0003). Splenic CD71-positive reticulocyte concentrations correlated with spleen weight in Pf (r = 1.0; p = .003). Retention rates of peripheral and splenic RBCs were correlated negatively with circulating RBC counts (r = -.69, p = .07 and r = -.83, p = .008, respectively). In conclusion, retention of mostly uninfected RBC in the spleen, leading to marked congestion of the red-pulp, was associated with splenomegaly and is the major mechanism of anemia in subjects infected with Plasmodium, particularly Pf.

Molecular profiling reveals features of clinical immunity and immunosuppression in asymptomatic P. falciparum malaria.

Clinical immunity to P. falciparum malaria is non-sterilizing, with adults often experiencing asymptomatic infection. Historically, asymptomatic malaria has been viewed as beneficial and required to help maintain clinical immunity. Emerging views suggest that these infections are detrimental and constitute a parasite reservoir that perpetuates transmission. To define the impact of asymptomatic malaria, we pursued a systems approach integrating antibody responses, mass cytometry, and transcriptional profiling of individuals experiencing symptomatic and asymptomatic P. falciparum infection. Defined populations of classical and atypical memory B cells and a TH2 cell bias were associated with reduced risk of clinical malaria. Despite these protective responses, asymptomatic malaria featured an immunosuppressive transcriptional signature with upregulation of pathways involved in the inhibition of T-cell function, and CTLA-4 as a predicted regulator in these processes. As proof of concept, we demonstrated a role for CTLA-4 in the development of asymptomatic parasitemia in infection models. The results suggest that asymptomatic malaria is not innocuous and might not support the induction of immune processes to fully control parasitemia or efficiently respond to malaria vaccines.

High-dimensional mass cytometry identifies T cell and B cell signatures predicting reduced risk of Plasmodium vivax malaria.

IFN-γ-driven responses to malaria have been shown to modulate the development and function of T follicular helper (TFH) cells and memory B cells (MBCs), with conflicting evidence of their involvement in the induction of antibody responses required to achieve clinical immunity and their association with disease outcomes. Using high-dimensional single-cell mass cytometry, we identified distinct populations of TH1-polarized CD4+ T cells and MBCs expressing the TH1-defining transcription factor T-bet, associated with either increased or reduced risk of Plasmodium vivax (P. vivax) malaria, demonstrating that inflammatory responses to malaria are not universally detrimental for infection. Furthermore, we found that, whereas class-switched but not IgM+ MBCs were associated with a reduced risk of symptomatic malaria, populations of TH1 cells with a stem central memory phenotype, TH17 cells, and T regulatory cells were associated with protection from asymptomatic infection, suggesting that activation of cell-mediated immunity might also be required to control persistent P. vivax infection with low parasite burden.

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

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.

Molecular surveillance over 14 years confirms reduction of Plasmodium vivax and falciparum transmission after implementation of Artemisinin-based combination therapy in Papua, Indonesia.

Genetic epidemiology can provide important insights into parasite transmission that can inform public health interventions. The current study compared long-term changes in the genetic diversity and structure of co-endemic Plasmodium falciparum and P. vivax populations. The study was conducted in Papua Indonesia, where high-grade chloroquine resistance in P. falciparum and P. vivax led to a universal policy of Artemisinin-based Combination Therapy (ACT) in 2006. Microsatellite typing and population genetic analyses were undertaken on available isolates collected between 2004 and 2017 from patients with uncomplicated malaria (n = 666 P. falciparum and n = 615 P. vivax). The proportion of polyclonal P. falciparum infections fell from 28% (38/135) before policy change (2004-2006) to 18% (22/125) at the end of the study (2015-2017); p<0.001. Over the same period, polyclonal P. vivax infections fell from 67% (80/119) to 35% (33/93); p<0.001. P. falciparum strains persisted for up to 9 years compared to 3 months for P. vivax, reflecting higher rates of outbreeding in the latter. Sub-structure was observed in the P. falciparum population, but not in P. vivax, confirming different patterns of outbreeding. The P. falciparum population exhibited 4 subpopulations that changed in frequency over time. Notably, a sharp rise was observed in the frequency of a minor subpopulation (K2) in the late post-ACT period, accounting for 100% of infections in late 2016-2017. The results confirm epidemiological evidence of reduced P. falciparum and P. vivax transmission over time. The smaller change in P. vivax population structure is consistent with greater outbreeding associated with relapsing infections and highlights the need for radical cure to reduce recurrent infections. The study emphasizes the challenge in disrupting P. vivax transmission and demonstrates the potential of molecular data to inform on the impact of public health interventions.

Use of a highly-sensitive rapid diagnostic test to screen for malaria in pregnancy in Indonesia.

BACKGROUND: The sensitivity of rapid diagnostic tests (RDTs) for malaria is inadequate for detecting low-density, often asymptomatic infections, such as those that can occur when screening pregnant women for malaria. The performance of the Alere™ Ultra-sensitive Malaria Ag Plasmodium falciparum RDT (uRDT) was assessed retrospectively in pregnant women in Indonesia. METHODS: The diagnostic performance of the uRDT and the CareStart™ Malaria HRP2/pLDH VOM (Plasmodium vivax, Plasmodium ovale and Plasmodium malariae) Combo RDT (csRDT) were assessed using 270 stored red blood cell pellets and plasma samples from asymptomatic pregnant women. These included 112 P. falciparum negative and 158 P. falciparum positive samples detected by a composite test (qPCR, LAMP, nPCR) as reference standard. Diagnostic indicators: sensitivity, specificity, negative predictive value (NPV), positive predictive value (PPV), diagnostic odds ratio (DOR) and the level of agreement (kappa) were calculated for comparison. RESULTS: Compared with the reference test, the uRDT had a sensitivity of 19.6% (95% CI 13.9-26.8) and specificity of 98.2% (93.1-99.7%). The csRDT was 22.8% (16.7-30.3) sensitive and 95.5% (89.4-98.3) specific for P. falciparum infections. Performance of the uRDT was non-significantly different to the csRDT (p = 0.169). RDT outcome was stratified by qPCR cycling threshold (Ct), and performance of the RDTs was found to be comparable across parasite loads. CONCLUSION: The uRDT performed similarly to the currently used csRDTs in detecting P. falciparum infections in asymptomatic pregnant women. In these settings, molecular diagnostics are currently the most sensitive for malaria.

Therapeutic Response to Dihydroartemisinin-Piperaquine for P. falciparum and P. vivax Nine Years after Its Introduction in Southern Papua, Indonesia.

Dihydroartemisinin-piperaquine (DHP) has been the first-line treatment of uncomplicated malaria due to both Plasmodium falciparum and Plasmodium vivax infections in Papua, Indonesia, since March 2006. The efficacy of DHP was reassessed to determine whether there had been any decline following almost a decade of its extensive use. An open-label drug efficacy study of DHP for uncomplicated P. falciparum and P. vivax malaria was carried out between March 2015 and April 2016 in Timika, Papua, Indonesia. Patients with uncomplicated malaria were administered supervised DHP tablets once daily for 3 days. Clinical and laboratory data were collected daily until parasite clearance and then weekly for 6 weeks. Molecular analysis was undertaken for all patients with recurrent parasitemia. A total of 129 study patients were enrolled in the study. At day 42, the polymerase chain reaction-adjusted efficacy was 97.7% (95% confidence intervals [CI]: 87.4-99.9) in the 61 patients with P. falciparum malaria, and 98.2% [95% CI: 90.3-100] in the 56 patients with P. vivax malaria. By day 2, 98% (56/57) of patients with P. falciparum and 96.9% (63/65) of those with P. vivax had cleared their peripheral parasitemia; none of the patients were still parasitaemic on day 3. Molecular analysis of P. falciparum parasites showed that none (0/61) had K13 mutations associated previously with artemisinin resistance or increased copy number of plasmepsin 2-3 (0/61). In the absence of artemisinin resistance, DHP has retained high efficacy for the treatment of uncomplicated malaria despite extensive drug pressure over a 9-year period.

Passively versus Actively Detected Malaria: Similar Genetic Diversity but Different Complexity of Infection.

The surveillance of malaria is generally undertaken on the assumption that samples passively collected at health facilities are comparable to or representative of the broader Plasmodium reservoir circulating in the community. Further characterization and comparability of the hidden asymptomatic parasite reservoir are needed to inform on the potential impact of sampling bias. This study explores the impact of sampling strategy on molecular surveillance by comparing the genetic make-up of Plasmodium falciparum and Plasmodium vivax isolates collected by passive versus active case detection. Sympatric isolates of P. falciparum and P. vivax were collected from a large community survey and ongoing clinical surveillance studies undertaken in the hypomesoendemic setting of Mimika District (Papua, Indonesia). Plasmodium falciparum isolates were genotyped at nine microsatellite loci and P. vivax at eight loci. Measures of diversity and differentiation were used to compare different patient and parasitological sample groups. The results demonstrated that passively detected cases (symptomatic) had comparable population diversity to those circulating in the community (asymptomatic) in both species. In addition, asymptomatic patent infections were as diverse as subpatent infections. However, a significant difference in multiplicity of infection (MOI) and percentage of polyclonal infections was observed between actively and passively detected P. vivax cases (mean MOI: 1.7 ± 0.7 versus 1.4 ± 1.4, respectively; P = 0.001). The study findings infer that, in hypomesoendemic settings, passive sampling is appropriate for molecular parasite surveillance strategies using the predominant clone in any given infection; however, the findings suggest caution when analyzing complexity of infection. Further evaluation is required in other endemic settings.