Enny Kenangalem

Background: Plasmodium falciparum and vivax are parasites responsible for most malaria cases globally. In areas where these species coexist, individuals gain protection from P vivax more rapidly, and important biological differences between species may affect immune responses. CD4 T cells are key drivers of immunity to malaria as effector and helper cells, with T follicular helper cells having key roles in antibody development. Comparative studies on CD4 T cell responses between these species are limited. Methods: We assessed CD4 T cells in adults with either P falciparum or P vivax malaria. Activation and proliferation of CD4 T cells were measured ex vivo, and functional capacity was determined by intracellular cytokine staining via flow cytometry. Results: The phenotype, activation, and proliferation of CD4 T cell subsets were largely comparable between species. However, within the peripheral T follicular helper (pTfh) cell compartment, there was some evidence for species-dependent activation, with relatively increased pTfh1 cells in P falciparum infection. Additionally, in P falciparum, increased IL-10 production was detected, including within IL-21–producing CD4 T cells. Conclusions: While activation and function of CD4 T cells in malaria are largely comparable, some species-dependent responses are detected within the pTfh-cell compartment that may affect antibody development.

Matryoshka RNA virus 1 (MaRNAV-1) is a bi-segmented and single-stranded RNA virus associated with Plasmodium vivax, a cause of human malaria. Little has been uncovered about the epidemiology and ecology of this virus since its discovery in 2019. To address this, we used a combination of primary and publicly available metatranscriptomic data to map the geographic distribution and host associations of MaRNAV-1. We detected this virus throughout Southeast Asia, in parts of South America, and, for the first time, in Oceania. Despite its broad distribution, MaRNAV-1 was found exclusively in metatranscriptomes containing P. vivax, suggesting that there is a specific virus-host relationship that has shaped the evolutionary history of this virus. We were unable to estimate the emergence date of the MaRNAV-1 lineage; however, phylogeographic mapping analysis suggested that MaRNAV-1 is widely dispersed throughout Southeast Asia. Our findings have both evolutionary and public health implications and can serve as the basis for future investigations in these fields.

Splenic rupture is a recognized complication of acute Plasmodium falciparum and Plasmodium vivax malaria, but the risk of splenic rupture in chronic asymptomatic infections is unknown. In Timika, Papua, Indonesia, we determined the proportion of PCR-detectable asymptomatic peripheral parasitemia in patients undergoing trauma-related splenectomy (2015–21) and found it was more than twice the proportion compared to a 2013 household survey of the general population (87.9% [29/33] vs 38.6% [697/1807]; P < .0001). Our findings suggest asymptomatic parasitemia with either P. falciparum or P. vivax is associated with splenic rupture following trauma, pointing toward an additional consequence of chronic infection in malaria-endemic areas.

Background: Plasmodium falciparum and P. vivax are parasites responsible for most malaria cases globally. In areas where these species co-exist, individuals gain protection from P. vivax more rapidly, and important biological differences between species may impact the immune response. CD4 T cells are key drivers of immunity to malaria, both as effector and helper cells, with T-follicular helper (Tfh) having key roles in antibody development. Comparative studies on CD4 T cell responses between these species are limited. Methods: We assessed CD4 T cells in adults with either P. falciparum or P. vivax malaria. Activation and proliferation of CD4 T cells were measured ex vivo , and functional capacity determined by intracellular cytokine staining by flow cytometry. Results: The phenotype, activation and proliferation of CD4 T cells and effector CD4 T cell subsets were comparable between species. However, within the peripheral (p)Tfh cell compartment, there was evidence for a skew towards pTfh1 cells in P. falciparum , and pTfh2 cells in P. vivax . Additionally, in P. falciparum , increased IL-10 production was detected, including within IL-21 producing CD4 T cells. Conclusion: While activation and function of CD4 T cells in malaria are largely comparable, some species-dependent responses are detected within the pTfh cell compartment that may impact antibody development.

Background: Dengue and malaria are major public health problems in Indonesia. Dengue is hyperendemic nationwide, while malaria remains endemic in specific regions, especially in eastern Indonesia. Timika in Central Papua province is a highly malaria-endemic area; however, this city is historically known as a low endemic area for dengue. With the increasing incidence of dengue in Papua, this study aimed to assess dengue prevalence, possible co-infection and to molecularly characterize the dengue virus (DENV) in patients suspected of having malaria. Methods: Archived samples of patients suspected of having malaria were screened for dengue using RT-PCR. Dengue serological and antigen detection was performed and whole genome sequencing was employed to determine the genetic characteristics of viruses. Detection of other arboviruses was also performed. Results: Among 119 patients, 30 (25.2%) were positive for dengue. Most samples (n=29) were infected by DENV-3, while one was positive for DENV-2. Dengue and malaria co-infections were found in six patients. Phylogenetic analysis classified DENV-2 as the Cosmopolitan genotype that is closely related to strains from the Indonesian city of Makassar, while DENV-3 was classified as Genotype I, which is closely related to a strain from Singapore. Conclusions: Dengue significantly contributes to febrile illness among patients suspected of having malaria in Timika, Central Papua. Virus importation from surrounding regions is likely to contribute to the dispersion of DENV into eastern parts of Indonesia. Our findings reveal the underestimation of this viral disease in a highly malaria-endemic area.

In humans, ∼0.1% to 0.3% of circulating red blood cells (RBCs) are present as platelet-RBC (P-RBC) complexes, and it is 1% to 2% in mice. Excessive P-RBC complexes are found in diseases that compromise RBC health (eg, sickle cell disease and malaria) and contribute to pathogenesis. However, the physiological role of P-RBC complexes in healthy blood is unknown. As a result of damage accumulated over their lifetime, RBCs nearing senescence exhibit physiological and molecular changes akin to those in platelet-binding RBCs in sickle cell disease and malaria. Therefore, we hypothesized that RBCs nearing senescence are targets for platelet binding and P-RBC formation. Confirming this hypothesis, pulse-chase labeling studies in mice revealed an approximately tenfold increase in P-RBC complexes in the most chronologically aged RBC population compared with younger cells. When reintroduced into mice, these complexes were selectively cleared from the bloodstream (in preference to platelet-free RBC) through the reticuloendothelial system and erythrophagocytes in the spleen. As a corollary, patients without a spleen had higher levels of complexes in their bloodstream. When the platelet supply was artificially reduced in mice, fewer RBC complexes were formed, fewer erythrophagocytes were generated, and more senescent RBCs remained in circulation. Similar imbalances in complex levels and senescent RBC burden were observed in humans with immune thrombocytopenia (ITP). These findings indicate that platelets are important for binding and clearing senescent RBCs, and disruptions in platelet count or complex formation and clearance may negatively affect RBC homeostasis and may contribute to the known risk of thrombosis in ITP and after splenectomy.

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 T _H2 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.

Background: In co-endemic areas there is a high risk of P. vivax recurrence in patients treated for P. falciparum malaria. Primauine radical cure has the potential to reduce P. vivax recurrences in patients presenting with P. falciparum as well as P. vivax malaria, but is undermined by poor adherence to the currently recommended 14 day regimen. We assessed the effectiveness of supervised primaquine radical cure in patients presenting with uncomplicated malaria. Methods: We conducted a cluster-randomised, controlled, open-label superiority trial in Papua, Indonesia. Twenty one clusters (village health post matched by location, size and malaria transmission) were assigned randomly (1:1) by a statistician independent to the trial to treat patients presenting with uncomplicated P. falciparum or P. vivax malaria with dihydroartemisinin-piperaquine plus either a supervised or unsupervised 14-day course of primaquine (0.5mg/kg per day). Patients were followed for 6 months and those representing with malaria were retreated with the same drug regimen. The primary outcome was the incidence risk and rate of P. vivax recurrent episodes over 6 months assessed in the intention to treat population. Findings: Between September 2016 and July 2018, 419 patients were enrolled. Overall the incidence risk of P. vivax recurrence in the 10 unsupervised clusters was 55.8% (95%CI, 32.3-81.8) compared to 29.7% (95%CI, 16.4-49.9) in the 11 supervised clusters (HR 0.23, 95%CI, 0.07-0.76, p=0.016). The incidence rate in unsupervised clusters was 859 (95%CI, 673-1,096) P. vivax infections per 1000 person years compared to 539 (95%CI, 390-747) in the supervised clusters (IRR 0.63, 95%CI, 0.42-0.94, p=0.025). The corresponding rates in the 224 patients presenting with P. falciparum malaria were 660 (95%CI, 446-977) and 346 (95%CI, 213-563); IRR 0.52 (95%CI, 0.28-0.98), p=0.043. There were no serious adverse events attributable to primaquine. Interpretation: In this area of moderate malaria transmission, supervision of primaquine radical cure reduced the risk of P. vivax recurrence and this was apparent for patients presenting with either P. falciparum or P. vivax malaria. Clinical Trial Registration Details: The trial was registered with the US National Library of Medicine, registered at ClinicalTrials.gov (NCT 02787070). Funding Information: The Bill and Melinda Gates Foundation, The Wellcome Trust and The Department of Foreign Affairs and Trade of the Australian Government. Declaration of Interests: No conflict of interests reported. Ethics Approval Statement: Ethical approval was obtained from the Human Research Ethics Committee of the Northern Territory Department of Health, Australia (HREC 15.2517) and the Health Research Ethics Committees of the University of Gadjah Mada, Indonesia (KE/FK/522/EC/2016).