Novel isothermal nucleic acid amplification method for detecting malaria parasites

Abstract

Malaria, a parasitic disease caused by Plasmodium spp. and transmitted by Anopheles mosquitoes, remains a major global health issue, with an estimated 249 million cases and 608,000 deaths in 2022. Rapid and accurate diagnosis and treatment are crucial for malaria control and elimination. However, limited access to sensitive molecular tests means that microscopic examination and rapid diagnostic tests (RDT) are the most used methods in endemic areas, despite their lower diagnostic accuracy. Therefore, there is a need for developing sensitive, simple, accurate, and rapid diagnostic tools suitable for field conditions. Herein, we aimed to explore the potential of the enzymatic recombinase amplification assay (ERA® Technology) as a remote laboratory test by evaluating and validating the GENEYE® ERA Plasmodium detection kit in Brazilian endemic areas. A cross-sectional cohort study was conducted between June and August of 2023 in the Brazilian Amazon. The study enrolled 323 participants residing in three malaria-affected regions: Cruzeiro do Sul and Mâncio Lima (Acre State) and Guajará (Amazonas State). The participants were tested for malaria by microscopy, rapid diagnostic tests (RDT), nested PCR (nPCR), quantitative real-time PCR (qPCR), and ERA. The sensitivity, specificity, and predictive values were assessed using nPCR as a gold standard. Plasmodium prevalence was 21.7%, 18.8%, 19.2%, 21.7%, and 21.7% by nPCR, microscopy, RDT, qPCR, and ERA respectively. Using nPCR as the standard, qPCR, and ERA showed a sensitivity of 100%. In comparison, microscopy and RDT showed a sensitivity of 87.1% and 88.6%, a negative predictive value (NPV) of 96.56 and 96.93, and kappa values of 0.91 and 0.92, respectively. For Plasmodium falciparum, the sensitivity of qPCR and ERA was 100% while the sensitivity of microscopy and RDT was 96.9% and 93.7%, and the NPV was 99.66 and 99.32, respectively. For Plasmodium vivax, only ERA showed the same sensitivity of nPCR. The sensitivity, NPV, and kappa values were 78.85%, 97.27, and 0.87 for qPCR and microscopy, and 84.21%, 97.94, and 0.9 for RDT. The data presented here show that the GENEYE® ERA Plasmodium detection kit offers a promising alternative to traditional malaria diagnostic methods. Its high sensitivity, specificity, fast processing time, and operational simplicity position it as a valuable point-of-care diagnostic tool, particularly in resource-limited and remote malaria-endemic areas. KEY POINTS: • GENEYE® ERA kit detects Plasmodium in under 25 min, no DNA purification needed. • The kit matches or exceeds the compared methods in sensitivity and specificity. • The kit is suitable for accurate testing in low-infrastructure, point-of-care settings.

Keywords: Plasmodium detection; Brazilian Amazon; Isothermal nucleic acid amplification; Malaria; Point-of-care; Rapid diagnostic tests.

Fig. 1

ERA® Technology’s DNA amplification diagram (adapted from GenDx Biotech, Suzhou, China). a Diagram of the ERA process. The ERA® Technology assay incorporates forward and reverse primers and engineered T4-UvsX to facilitate primer binding and prevent contamination. It also includes T4 ssDNA-binding proteins to stabilize the ssDNA and Bsu DNA polymerase 2.0 (lacking 5′−3′ exonuclease activity) to amplify the primer-bound strands. The amplification process is shown step-by-step in the figure. b Exonuclease-based detection of the ERA product. An exo-probe, flanked by a quencher (Q) and a fluorophore (F), binds to the amplified product. The probe contains a tetrahydrofuran (THF) residue, cleaved by exonuclease III, releasing the fluorophore from the quencher. The blue circle with the letter B represents a 3′ blocking group. The resulting fluorescent signal indicates the presence of the target nucleic acid sequence

Fig. 2

Brazil map showing studied areas in the spotlight. Areas of malaria transmission in Brazil and studied areas according to the Annual Parasitary Index (API, number of autochthonous cases per 1000 inhabitants. Very low API indicates that there are less than 1 case/1000 inhabitants, low API indicates that there are less than 10 cases/1000 inhabitants, medium API indicates 10–49.9 cases/1000 inhabitants and high API more than 50 cases/1000 inhabitants (Malaria-Brasil 2023)

Fig. 3

Schematic workflow of GENEYE® ERA Plasmodium detection kit. WB: whole blood, A: Buffer A, M: Buffer M, L: sample lysate. 1. Pre-warm the device by heating the bath to 40 °C; 2. Turn on and connect the isothermal device to the app; 3. Organize and prepare all necessary reagents; 4. Add 20 µL of whole blood to 2 mL of buffer A; 5. Carefully homogenize the tube and let it incubate at room temperature for 5 min; 6. Add 40 µL of lysate to the bottom of the piston tube, followed by 10 µL of buffer M to the piston tube’s inner bottom wall; 7. After closing the piston tube cap tightly, add 50 µL of buffer M to the top cavity of the piston tube and mix it carefully by hand; 8. Place the piston tube in the dry bath at 40 °C for a 15-min incubation period; 9. Press the pistons to mix the buffer M and the bottom pre-mix, then remove the top of the piston tube; 10. Insert the piston tube into the GENEYE® Mini Isothermal ERA device; 11. After 5 min, check the fluorescence readings on the app to determine if the result is “negative” or “positive”

Fig. 4

Visual representation comparing the prevalence rates determined by the malaria diagnostic tools: nPCR, GENEYE® ERA Plasmodium detection kit, qPCR, microscopy, and RDT