Agatha Mia Puspitasari

Summary: Backgroun: Persistent transmission from relapsing Plasmodium vivax infections threatens malaria elimination programs in the Asia-Pacific and Americas. Tools to identify people at risk of relapse are urgently required. We aimed to validate a panel of eight P. vivax serological biomarkers for predicting future relapses. Methods: In this observational study, soldiers returning from malaria-endemic Papua to non-endemic East Java, Indonesia, were screened at enrolment using antibody measurement (Luminex) and trained random forest classification algorithms, then followed for 6 months. Active case detection was performed fortnightly by microscopy. Algorithms classified soldiers as recently infected (last nine months) and thus at risk of relapse, based on anti-vivax antibody measurements at enrolment. Findings: Between December 2018 and July 2022, 592 soldiers were enrolled, with 553 completing follow-up; 119 experienced a P. vivax relapse. Of these, 102 were correctly classified as at risk of relapse at enrolment, corresponding to 86% sensitivity and 86% specificity, with an AUC of 0.92. Interpretation: P. vivax serological biomarkers can identify people at risk of relapse with high sensitivity and specificity and could be used as a novel public health intervention, P. vivax serological testing and treatment (PvSeroTAT), to reduce relapse-driven transmission. Funding: Bill and Melinda Gates Foundation (OPP1180981) funded the study trial. LJR, RJL and IM received National Health and Medical Research Council Fellowships (#2017630, #1173210, #2016726). RJL receives salary support from the Victorian Government as a veski FAIR Fellow and from the Sylvia and Charles Viertel Charitable Foundation as a Viertel Senior Medical Research Fellow. LJR, RJL and IM receive funding from NHMRC Synergy grant (NHMRC #2018654) and are part of the Australian Centre of Research Excellence in Malaria Elimination (NHMRC #2024622). Conflict of interest: IM, MTW and RJL are named inventors on a patent describing P. vivax serological exposure markers (PCT/US17/67926). All other authors declare no competing interests. Research in context: Evidence before this study: Relapses from long-lasting liver stages (i.e. hypnozoites) account for 80-90% of all P. vivax blood-stage infections ^1,2 and it has been well understood that regional P. vivax elimination will be challenging without directly attacking this hidden parasite reservoir. Doing this efficiently will require novel diagnostic tests that can detect individuals at risk of relapse (as described in a recently published WHO preferred product characteristics (PPC) ³ . None of the current malaria diagnostic tests can detect hypnozoite carriers and/or identify people at risk of relapse. A previous study showed that antibodies to a validated panel of 8 P. vivax antigen markers can detect P. vivax infection within the previous 9 months with 80% sensitivity and 80% specificity, and identify people with significantly higher risk of experiencing future P. vivax infections. ⁴ These findings suggested that these serological exposure markers (SEMs) may be a proxy for the presence of hypnozoites in the liver and thus act as biomarkers for future relapse risk. Added value of this study: By following two cohorts of soldiers between 2018 to 2021, the present study has been able to confirm that antibodies to 8 P. vivax SEMs are able to identify individuals who experienced a P. vivax relapse during 6 months following their return from a malaria endemic region in Papua, Indonesia to their non-endemic base in Java with high performance (86% sensitivity, 86% specificity, AUC: 0.92). This proves that P. vivax SEMs are indeed markers of future relapse risk and thus valid surrogate markers for hypnozoite carriage. Implications of all the available evidence: The validated P. vivax sero-diagnostic assay is the first ever diagnostic to fulfil the requirements outlined in the recently published WHO PPC for tests to identify individuals at risk of P. vivax relapse. This makes the test suitable for use in both public health interventions, such as serological testing & treatment (PvSeroTAT), where serology is used to identify people at risk of relapse to guide radical cure, and for sero-surveillance for risk stratification and monitoring and evaluation of ongoing elimination programs. Trials of both uses are currently ongoing in different vivax endemic areas of the Asia-Pacific, South America and Africa.

Objective: Malaria and dengue are two important infectious diseases in Indonesia, but information about their co-infections is still limited. This study aimed to describe malaria–dengue co-infections in Timika, Papua, including the clinical manifestations and genetic characteristics of the infecting dengue virus (DENV). Methods: Sixty-nine microscopy-confirmed malaria patient samples from Timika, a malaria-highly endemic region in Central Papua province, Indonesia collected in 2022 were screened for DENV infection using qRT-PCR. Malaria speciation was performed using qRT-PCR. The whole genomes of the viruses were sequenced using MinION Oxford Nanopore Technology, and the phylogenetic analysis was inferred to determine the origin, genotype, and genetic characteristics of the viruses. Results: Out of 69 malaria-positive samples, three patients (4.3%) were co-infected by DENV-2 (1 patient) and DENV-4 (2 patients). The infecting parasites were P. vivax, P. falciparum , and P. malariae as determined using qRT-PCR. The whole genomes of the infecting DENV-4 were successfully sequenced and phylogenetic analysis revealed the genotype of the virus as Genotype II, which were closely related to DENV strains from Makassar city in South Sulawesi province, Indonesia. Conclusions: This study demonstrates the occurrence of malaria and dengue co-infections in Timika, Central Papua, Indonesia, with a prevalence of 4.3%. The co-infection poses a unique clinical and diagnostics challenges due to overlapping symptoms and potential complications. This finding highlights the diagnostic difficulties, clinical presentations, and future possibilities for such cases. The genomic data of DENV obtained from these cases provide new information about virus circulation in Indonesia and support the importance of combining clinical and molecular approaches for surveillance.

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.

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: A very large biomass of intact asexual-stage malaria parasites accumulates in the spleen of asymptomatic human individuals infected withPlasmodium vivax. The mechanisms underlying this intense tropism are not clear. We hypothesised that immature reticulocytes, in whichP.vivaxdevelops, 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 toP.vivaxandPlasmodium falciparumundergoing 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 splenicPlasmodiuminfection (7P.vivax, 13P.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-stageP.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-bodyP.vivaxbiomass. More reticulocytes were in contact with sinus lumen endothelial cells inP.vivax- than inP.falciparum-infected spleens. Histological analyses revealed 96% ofP.vivaxrings/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 splenicP.vivax-infected erythrocytes of all asexual stages accumulate in the same splenic compartments, suggesting the existence of a cryptic endosplenic lifecycle in chronicP.vivaxinfection. Findings provide insight intoP.vivax-specific adaptions that have evolved to maximise survival and replication in the spleen.