Submicroscopic burden of zoonoticP. knowlesimalaria on Mursala Island andP. falciparumandP. vivaxtransmission in mainland North Sumatra, Indonesia

Abstract Accurate molecular tools are essential for estimating zoonotic malaria transmission in Southeast Asia. This study applied ultrasensitive reverse-transcriptase real-time PCR to detect zoonotic malaria in febrile patients from eight mainland health facilities and Mursala Island, spanning three districts in North Sumatra, Indonesia. Among 64 participants on Mursala, 7 (10.9%) had confirmed Plasmodium knowlesi symptomatic infections, including two submicroscopic infections associated with severe anaemia. All were negative by microscopy and pan-pLDH rapid diagnostic tests. No P. knowlesi infections were identified among 947 participants from mainland sites; PCR detected malaria in 34%, including P. vivax (17.5%) and P. falciparum (7.5%). Of these, 30% were submicroscopic infections. No P. cynomolgi infections were identified. P. knowlesi transmission is low in North Sumatra, however, may cause serious disease. Molecular diagnostics remain crucial for identifying zoonotic malaria and should be integrated into surveillance systems to inform public health control measures. Competing Interest Statement The authors have declared no competing interest. Funding Statement This work was supported by the Australian Centre for International Agricultural Research and the Department of Foreign Affairs and Trade, Australian Government (LS/2018/214), and the National Health and Medical Research Council, Australia (fellowships to NMA [1042072], MJG [1138860]). Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethics committee/IRB of Universitas Sumatera gave ethical approval for this work (No. 179/TGL/KEPK FK-USU-RSUPHAM/2019). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Data Availability All data produced in the present study are available upon reasonable request to the authors.