Diagnostic approaches to the detection of zoonotic Plasmodium knowlesi infections and the role of neutrophil activation in severe malaria

Abstract

Plasmodium knowlesi, a macaque parasite transmitted by the Anopheles Leucosphyrus group of mosquito vectors, is the primary cause of zoonotic malaria in Southeast Asia. The emergence of P. knowlesi poses a significant public health issue due to its potential for severe disease with fatality rate similar to P. falciparum, particularly when diagnosis and treatment are delayed. This thesis comprises three published studies that address the WHO-identified research priority of developing and evaluating rapid and sensitive diagnostic tools for P. knowlesi, to improve both accurate public health reporting and clinical outcomes. The fourth publication focused on neutrophil-mediated pulmonary complications in knowlesi malaria. The first publication in this thesis compared the diagnostic performance of two real-time multiplex polymerase chain reaction (PCR) assays (QuantiFast™ and abTES™) utilized by a state reference laboratory in Malaysia for zoonotic and human-only Plasmodium species confirmation and surveillance reporting. Both assays utilizeP. knowlesi-specific primers and probes at detection limits of 20 and ≤0.125 parasites/µL respectively, with QuantiFast™ achieving comparable accuracy despite costing nearly a third less than abTES™. Findings highlight necessity for regional harmonisation of validated methods for zoonotic malaria surveillance, in this context, QuantiFast™ for first-line molecular detection, followed by abTES™ for confirmation as required. The second publication evaluated the diagnostic accuracy and limit of detection (LOD) of ten World Health Organization (WHO)-prequalified, antigen-based lateral flow rapid diagnostic tests (RDTs) using for P. knowlesi detection. Selected RDTs were assessed against 127 PCR-confirmed P. knowlesi cases and malaria-negative controls, and LODs measured using cultured P. knowlesi (PkA1-H.1). The best performing Plasmodium-genus parasite lactate dehydrogenase (pan-pLDH) and P. vivax (Pv)-pLDH tests yielded sensitivities of 87.0% and 92.0%, and limits of detection of 25 and 49 parasites/μL respectively for P. knowlesi, with no false-positives in P. falciparum-specific channels. Results of top-performing RDTs demonstrated improved performance for P. knowlesi detection compared to previous RDT-based studies, with acceptable sensitivity for field implementation where microscopy is unavailable. The third publication assessed a novel haemozoin-based magneto-optical detection platform for field applicability in detecting 243 PCR-confirmed P. knowlesi cases. Despite sensitivity of 94.6% and an LOD of 33 parasites/µL, improvements in Plasmodium species-specific differentiation, reduced cost of tests, and higher throughput may improve feasibility for widespread implementation in co-endemic regions of P. knowlesi and other human malaria species. Haemozoin in neutrophils is an established prognostic marker for severe falciparum malaria, however, neutrophil activation in severe knowlesi malaria remains unexplored. The fourth publication described elevated plasma concentrations of neutrophil activation markers and increased circulating neutrophil extracellular traps in 232 P. knowlesi malaria patients. Elevated neutrophil elastase levels in patients with severe knowlesi malaria were demonstrated in those with clinical markers of acute lung injury, suggesting a role for neutrophils in the pathogenesis of pulmonary complications. The findings included in my thesis have addressed key evidence gaps in clinical diagnosis and regional surveillance approaches for P. knowlesi infections and advanced our knowledge in the immunopathogenesis of knowlesi malaria, potentially guiding treatment strategies in diverse settings across Southeast Asia.

Date of Award	3 May 2024
Original language	English
Awarding Institution	Charles Darwin University (CDU)
Supervisor	Bridget Barber (Supervisor), Nicholas Anstey (Supervisor), Matthew Grigg (Supervisor) & Steven Kho (Supervisor)