A Pan Plasmodium lateral flow recombinase polymerase amplification assay for monitoring malaria parasites in vectors and human populations

Abstract

Robust diagnostic tools and surveillance are crucial for malaria control and elimination efforts. Malaria caused by neglected Plasmodium parasites is often underestimated due to the lack of rapid diagnostic tools that can accurately detect these species. While nucleic-acid amplification technologies stand out as the most sensitive methods for detecting and confirming Plasmodium species, their implementation in resource-constrained settings poses significant challenges. Here, we present a Pan Plasmodium recombinase polymerase amplification lateral flow (RPA-LF) assay, capable of detecting all six human infecting Plasmodium species in low resource settings. The Pan Plasmodium RPA-LF assay successfully detected low density clinical infections with a preliminary limit of detection between 10-100 fg/µl for P. falciparum. When combined with crude nucleic acid extraction, the assay can serve as a point-of-need tool for molecular xenomonitoring. This utility was demonstrated by screening laboratory-reared Anopheles stephensi mosquitoes fed with Plasmodium-infected blood, as well as field samples of An. funestus s.l. and An. gambiae s.l. collected from central Africa. Overall, our proof-of-concept Pan Plasmodium diagnostic tool has the potential to be applied for clinical and xenomonitoring field surveillance, and after further evaluation, could become an essential tool to assist malaria control and elimination.

Fig. 1

(A) Inclusivity assessment for the Pan Plasmodium RPA-LF assay across all 6 human infecting Plasmodium species. P. falciparum (Pf), P. vivax (Pv), P. malariae (Pm), P. knowlesi (Pk), P. ovale curtisi (Poc), P. ovale wallikeri (Pow). (B) Limit of detection (LoD) assessment for the Pan Plasmodium RPA-LF assay for P. falciparum. Lateral flow cassettes have been cropped to remove the loading port. Lane C (C) represents the flow-check line and Lane 2 (2) detects the FAM/Biotin labelled amplicons generated via the Pan Plasmodium RPA-LF assay.

Fig. 2

A subset of the P. falciparum and P. vivax clinical isolates screened using the Pan Plasmodium RPA-LF assay, including the negative P. vivax isolates (V122). Lateral flow cassettes have been cropped to remove the loading port. Lane C (C) represents the flow-check line and Lane 2 (2) detects the FAM/Biotin labelled amplicons generated via the Pan Plasmodium RPA-LF assay.

Fig. 3

Screening of eight infected-blood-fed mosquitoes (AS1-8) and two uninfected An. stephensi mosquitoes (ASN1, ANS2), provided by the Human Malaria Transmission facility. The positive control (PC) and negative control (NC) consisted of P. falciparum and water, respectively. Lateral flow cassettes have been cropped to remove the loading port. Lane C (C) represents the flow-check line and Lane 2 (2) detects the FAM/Biotin labelled amplicons generated via the Pan Plasmodium RPA-LF assay.

Fig. 4

A subset of single (AC) and pools (AP) of infected-blood-fed Anopheles spp mosquitoes screening using the Pan Plasmodium RPA-LF assay following crude extraction. Lateral flow cassettes have been cropped to remove the loading port. Lane C (C) represents the flow-check line and Lane 2 (2) detects the FAM/Biotin labelled amplicons generated via the Pan Plasmodium RPA-LF assay.