Development and evaluation of PlasmoPod: A cartridge-based nucleic acid amplification test for rapid malaria diagnosis and surveillance

Abstract

Malaria surveillance is hampered by the widespread use of diagnostic tests with low sensitivity. Adequate molecular malaria diagnostics are often only available in centralized laboratories. PlasmoPod is a novel cartridge-based nucleic acid amplification test for rapid, sensitive, and quantitative detection of malaria parasites. PlasmoPod is based on reverse-transcription quantitative polymerase chain reaction (RT-qPCR) of the highly abundant Plasmodium spp. 18S ribosomal RNA/DNA biomarker and is run on a portable qPCR instrument which allows diagnosis in less than 30 minutes. Our analytical performance evaluation indicates that a limit-of-detection as low as 0.02 parasites/μL can be achieved and no cross-reactivity with other pathogens common in malaria endemic regions was observed. In a cohort of 102 asymptomatic individuals from Bioko Island with low malaria parasite densities, PlasmoPod accurately detected 83 cases, resulting in an overall detection rate of 81.4%. Notably, there was a strong correlation between the Cq values obtained from the reference RT-qPCR assay and those obtained from PlasmoPod. In an independent cohort, using dried blood spots from malaria symptomatic children living in the Central African Republic, we demonstrated that PlasmoPod outperforms malaria rapid diagnostic tests based on the PfHRP2 and panLDH antigens as well as thick blood smear microscopy. Our data suggest that this 30-minute sample-to-result RT-qPCR procedure is likely to achieve a diagnostic performance comparable to a standard laboratory-based RT-qPCR setup. We believe that the PlasmoPod rapid NAAT could enable widespread accessibility of high-quality and cost-effective molecular malaria surveillance data through decentralization of testing and surveillance activities, especially in elimination settings.

Fig 1. DiaxxoPCR and PlasmoPod setup for rapid molecular malaria detection.

A) The DiaxxoPCR instrument is a pyramid-shaped stand-alone device for rapid qPCR cycling and fluorescence acquisition. (B) The PlasmoPod assay is based on a single-use cartridge which is pre-loaded with all qPCR reagents in dried form. (C) The PlasmoPod laboratory setup consisting of the diaxxoPCR device, a heatblock, a pipette and PlasmoPod cartridges. (D) Finger prick blood is sampled and stored as dried blood spots (DBS). (E) The setup for rapid NA extraction from DBS. (F) The NA are amplified and signals analysed using the diaxxoPCR device and its integrated analysis software. Images A and B are republished from diaxxo AG under a CC BY license, with permission from Dr Michele Gregorini (who is an author of this manuscript), original copyright [diaxxo AG, 2020–2022]. The granted permission to the manuscript has been uploaded. Partially created with Biorender.com.

Fig 2. Analytical performance evaluation of PlasmoPod.

(A) DiaxxoPCR derived delta fluorescence and maximum slope after amplification with nucleic acid from different pathogens. Lines indicate the delta fluorescence positivity cutoff (vertical) and maximum slope positivity cutoff (horizontal). (B) Cq value of PlasmoPod versus dilutions of NAs extracted from culture-derived ring-stage synchronized P. falciparum parasites. Sample without amplification are colored in red.

Fig 3. Analysis of asymptomatic malaria cohort.

(A) Correlation of of Cq valuesvales obtained from reference RT-qPCR run on the Biorad CFX96 instrumentintrument and PlasmoPod run on diaxxoPCR. Samples negative for PlasmoPod were assigned a Cq value of -1. (B) Detection probability for PlasmoPod modelled based on reference Cq values. The grey area represents the 95% confidence interval. (C) Detection rate of PlasmoPod stratified by age group. (D) Cq values stratified by age group.

Fig 4. Clinical and parasitological characterization of study population.

(A) Correlation of parasite density assessed by the PfRNR2 assay and thick blood smear microscopy. (B) Modelled distribution of parasite densities measured by PfRNR2 assay and highlighted for high- and moderate-density infections. (C) Hematological parameters, including red blood cell counts (RBC), Hemoglobin levels (Hb), Thrombocyte counts (PLT) and white blood cell counts (WBC) compared between malaria negative children and children with high- and moderate-density malaria infections.

Fig 5. Quantification of P. falciparum parasites using PlasmoPod.

(A) Correlation of Cq values derived from PlasmoPod and reference (RT)-qPCR assays based on Plasmodium spp. 18S ribosomal DNA and RNA (left panel) and the P. falciparum ribonucleotide reductase R2_e2 assays (right panel). (B) Correlation between PlasmoPod and thick blood smear microscopy for quantification of P. falciparum parasites. The grey color represents the 95% confidence interval.