Rintis Noviyanti

In chronic Plasmodium vivax (Pv) infection, the spleen accounts for over 98% of total‐body parasite biomass. Whether splenic tropism also occurs in acute infection and how the spleen influences pathogenesis have not been systematically explored. In Papua, Indonesia, we compared clinical and hematology data in 24 spleen‐intact and 25 previously splenectomized patients with acute uncomplicated vivax malaria. Plasma levels of Pv lactate dehydrogenase (PvLDH) and markers of intravascular haemolysis, inflammation, endothelial activation and neutrophil activation were measured by ELISA. Circulating parasitaemia was four times higher in splenectomized compared to spleen‐intact patients but total‐body Pv biomass (PvLDH) was three times lower in patients without a spleen. Parasite staging and greater organ‐specific symptoms suggest parasite redistribution in the absence of a spleen. Linear regression modeling adjusting for circulating parasitaemia, age, sex and fever duration demonstrated an 8.1‐fold higher PvLDH concentration in spleen‐intact patients, indicating a splenic biomass accounting for 89% of total‐body parasites. Giemsa‐based splenic histology in an untreated patient splenectomized during acute vivax malaria revealed a spleen‐to‐blood biomass ratio of 10.7. In spleen‐intact patients, splenic biomass correlated strongly with markers of disease intensity, endothelial activation and systemic inflammation, whereas circulating parasitaemia correlated weakly or not at all. Compared to spleen‐intact patients, cell‐free hemoglobin, endothelial activation and systemic inflammation were higher in splenectomized patients while inflammasome‐dependent responses were lower. In conclusion, Pv is predominantly an infection of the spleen in acute clinical vivax malaria. While the size of this hidden population correlates with disease intensity, the spleen likely modulates inflammatory pathways and heme‐associated pathology.

Hemoglobin (Hb) testing is widely used to inform clinical care and assess population health. A World Health Organization (WHO)-UNICEF expert group recently recommended use of venous blood tested using an automated hematology analyzer as best practice for Hb measurement in population surveys. Recently updated WHO guidelines recommend the same methods for both populations and individuals. However, in low-resource settings where these methods are often inaccessible, the performance and clinical utility of simpler methods, defined by their accuracy for key clinical decisions and impact on patient management, should be considered. We discuss Hb test performance requirements for clinical contexts as opposed to population surveys, and consider acceptability criteria specific to a test’s context and intended use. Using the example of malaria case management and radical cure, we consider the clinical risk/benefit of the incidental Hb measurement provided on a point-of-care (POC) test for glucose-6-phosphate dehydrogenase (G6PD) deficiency (the STANDARD G6PD Test), which is required to inform optimal malaria radical cure. We review evidence from clinical evaluations, where Hb was measured in venous and capillary blood using the HemoCue 201+ and the STANDARD G6PD Test, and compared to venous Hb results from an automated hematology analyzer. If Hb was not measured, radical cure could be incorrectly prescribed or withheld in more individuals with severe anemia, relative to treatment errors that could be made if any POC Hb test method were used. Overall agreement with the gold-standard Hb measure varied from 98.4% to 99.5% for identifying severe anemia. In the absence of Hb POC testing, diagnosing anemia based on clinical signs is unlikely to improve these results. We highlight the importance of evaluating the acceptable performance of POC tests by considering clinical risks/benefits specific to the intended use and health system context to improve case management where alternate solutions are unavailable or unaffordable.

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.

Plasmodium vivax presents a significant obstacle to malaria elimination due to its capacity to form dormant liver-stage hypnozoites that can cause relapses. Universal radical cure, which administers hypnozoite-targeting treatment to patients with P. falciparum malaria living in co-endemic areas, has potential to reduce P. vivax relapses. However, its implementation is hindered by the lack of a diagnostic tool for detecting hypnozoite-carriage. P. vivax serological exposure markers (SEMs) were evaluated as a screening tool in P. falciparum patients for predicting risk of P. vivax microscopy-detected episodes over the following 63 days. Analysis was performed using samples from participants in the PRIMA study from Ethiopia, Indonesia, and Bangladesh (NCT03916003). An existing random forest serological classification algorithm was used and evaluated for sensitivity and specificity, then further optimized by re-training a study-specific model. IgG antibodies were measured in 244 P. falciparum participant samples, of which 22 had a vivax microscopy-detected episode during follow-up. SEMs showed high sensitivity (82%) but low specificity (27%), likely due to sustained antibody responses in moderate-to-high transmission areas or undetected sub-microscopic P. vivax infections during follow-up. A study-specific algorithm increased specificity to 68%, but with a corresponding drop in sensitivity to 68%. Although P. vivax SEMs showed limited suitability for guiding radical cure in P. falciparum patients in this study (where the outcome was microscopy-detected vivax episodes), they could play a valuable role in building community trust and acceptance of universal radical cure when supported by strong communication and implementation strategies.

West Nile virus (WNV) is a mosquito-borne flavivirus primarily transmitted by Culex species and maintained in a bird–mosquito–bird cycle. Though WNV is not considered endemic to Indonesia, sporadic molecular and serological findings suggest possible under-recognized circulation. To examine this possibility, we conducted a cross-sectional serosurvey of 569 archived serum and plasma samples collected between 2021 and 2023 from non-febrile individuals, including blood donors in Papua and community-based participants in Kalimantan. Samples were initially screened using a commercial WNV IgM Enzyme Linked Immunosorbent Assay (ELISA), IgM-positive and equivocal samples were further tested with IgM ELISAs against closely related flaviviruses, including dengue virus (DENV), Japanese encephalitis virus (JEV), and Zika virus (ZIKV). WNV-reactive IgM was initially detected in 9 (1.58%) samples, with 7 (1.23%) additional equivocal results. After additional testing, the WNV IgM seroprevalence was 0.35%, while 1.76% showed cross-reactivity to other flaviviruses. These cases spanned regions including North Kalimantan and Papua, where evidence of WNV-related exposure was previously reported. Detection of WNV-reactive IgM in asymptomatic individuals suggests potential silent or undetected circulation of WNV in Indonesia. Given diagnostic cross-reactivity and IgM persistence, further studies using molecular tools, plaque reduction neutralization test (PRNT), IgG testing, and entomological data are needed to clarify WNV epidemiology and inform surveillance strategies in the region.

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: 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.

Marsha S. Santoso, M.D, M.I.P.H., Eggi Arguni, M.D, Ph.D., Bunga Rana, M.Sc., Mercy E. Adiniko, M.Sc., Dionisius Denis, M.Sc., Endah Supriyati, M.Sc., Citra Indriani, M.D, M.P.H., Leily Trianty, Ph.D., Riris Andono Ahmad, M.D, Ph.D., Rintis Noviyanti, Ph.D., and R. Tedjo Sasmono, Ph.D.. Ann Lab Med -0001;0:. https://doi.org/10.3343/alm.2024.0558