Serological Markers Predict Plasmodium vivax Relapses in Returning Indonesian Soldier Cohorts

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.