Detection of P. malariae using a new rapid isothermal amplification lateral flow assay

Abstract

Background: While Plasmodium falciparum and Plasmodium vivax cause the majority of malaria cases and deaths, infection by Plasmodium malariae and other Plasmodium species also causes morbidity and mortality. Current understanding of these infections is limited in part by existing point-of-care diagnostics that fail to differentiate them and have poor sensitivity for low-density infections. Accurate diagnosis currently requires molecular assays performed in well-resourced laboratories. This report describes the development of a P. malariae diagnostic assay that uses rapid, isothermal recombinase polymerase amplification (RPA) and lateral-flow-strip detection.

Methods: Multiple combinations of custom RPA primers and probes were designed using publicly available P. malariae genomic sequences, and by modifying published primer sets. Based on manufacturer RPA reaction conditions (TwistDx nfo kit), an isothermal assay was optimized targeting the multicopy P. malariae 18S rRNA gene with 39 °C incubation and 30-min run time. RPA product was visualized using lateral strips (FAM-labeled, biotinylated amplicon detected by a sandwich immunoassay, visualized using gold nanoparticles). Analytical sensitivity was evaluated using 18S rRNA plasmid DNA, and clinical sensitivity determined using qPCR-confirmed samples collected from Tanzania, Ethiopia, and the Democratic Republic of the Congo.

Results: Using 18S rRNA plasmid DNA, the assay demonstrates a detection limit of 10 copies/µL (~ 1.7 genome equivalents) and 100% analytical specificity. Testing in field samples showed 95% clinical sensitivity and 88% specificity compared to qPCR. Total assay time was less than 40 min.

Conclusion: Combined with simplified DNA extraction methods, the assay has potential for future field-deployable, point-of-care use to detect P. malariae infection, which remains largely undiagnosed but a neglected cause of chronic malaria. The assay provides a rapid, simple readout on a lateral flow strip without the need for expensive laboratory equipment.

Keywords: P. malariae; Diagnostics; Isothermal nucleic acid amplification; Lateral flow; Point-of-care testing; RPA; Rapid test; Recombinase polymerase amplification.

Fig. 1

Plasmodium malariae lateral-flow based RPA assay schematic. (1) Reaction components include unlabelled forward and 5′-biotinylated reverse primers, 5′-FAM labelled probe with an abasic residue and 3′ blocker, endonuclease IV enzyme (nfo), and template DNA. (2) RPA reaction proceeds via primer-recombinase-SSB complex de-looping, nfo cleavage, polymerase extension that results in FAM- and biotin-labelled double-stranded amplicons. Exponential amplification occurs during 20-min isothermal incubation at 39 °C. (3) Lateral flow strips with a band containing anti-FAM antibodies are loaded with RPA product diluted in buffer containing streptavidin-conjugated gold nanoparticles. (4) Detection of labelled RPA product immobilized on the lateral flow strip is performed by visual inspection using the naked eye. Total reaction time is approximately 35 min. Figure made using BioRender

Fig. 2

Assay workflow and timeline. Sample collection, DNA extraction (and thawing of DNA and reagents, if necessary) times vary by method. Newer methods enable rapid extraction in ≤ 10 min (e.g., QuickExtract DNA, Biosearch Technologies, Hoddesdon, UK), but at higher cost, whereas conventional Chelex extraction can require up to 24 h at lower cost. Incubation temperature can be varied, though 39 °C was optimal for the assay. Visualization of positive results is often possible in < 2 min but reported at 5 min to standardize assay output. Figure made using BioRender

Fig. 3

Sensitivity and specificity of the P. malariae RPA-lateral flow (RPA-LF) assay. A Analytical sensitivity and specificity determined using serially diluted 18S rRNA plasmid copies/µL (equivalent to approximately 1700–0.17 genome equivalents/µL). B Example lateral flow read-out by species (Pf, P. falciparum; Pv, P. vivax; Po, P. ovale spp.; Pm, P. malariae; NTC, no-template control). C Clinical sensitivity determined using 21 Tanzania field samples with qPCR-confirmed P. malariae infection with a range of qPCR-determined parasite densities (log₁₀). One low-density case was missed (*). Three cases of Pm + Pf co-infection were included (^); the parasite density indicated for these Pm + Pf cases refers only to Pm. D Clinical specificity determined using Tanzania and Ethiopia field samples with qPCR-confirmed P. falciparum, P. ovale, and P. vivax