A Novel Malaria Pf/Pv Ab Rapid Diagnostic Test Using a Differential Diagnostic Marker Identified by Network Biology

Abstract

Rapid diagnostic tests (RDTs) can detect anti-malaria antibodies in human blood. As they can detect parasite infection at the low parasite density, they are useful in endemic areas where light infection and/or re-infection of parasites are common. Thus, malaria antibody tests can be used for screening bloods in blood banks to prevent transfusion-transmitted malaria (TTM), an emerging problem in malaria endemic areas. However, only a few malaria antibody tests are available in the microwell-based assay format and these are not suitable for field application. A novel malaria antibody (Ab)-based RDT using a differential diagnostic marker for falciparum and vivax malaria was developed as a suitable high-throughput assay that is sensitive and practical for blood screening. The marker, merozoite surface protein 1 (MSP1) was discovered by generation of a Plasmodium-specific network and the hierarchical organization of modularity in the network. Clinical evaluation revealed that the novel Malaria Pf/Pv Ab RDT shows improved sensitivity (98%) and specificity (99.7%) compared with the performance of a commercial kit, SD BioLine Malaria P.f/P.v (95.1% sensitivity and 99.1% specificity). The novel Malaria Pf/Pv Ab RDT has potential for use as a cost-effective blood-screening tool for malaria and in turn, reduces TTM risk in endemic areas.

Keywords: Blood screening; MSP1; Malaria antibody test; Transfusion-transmitted malaria.

Figure 1

Protein-protein interaction (PPI) network of Plasmodium falciparum. (A) Each node (shown by Black square) represents each protein and links represent the interconnection among nodes. (B) Distribution of nodes based on their degrees in the PPI network. R = 0.987, R² = 0.938

Figure 2

(A) Schematic drawing of decomposing PPI network by k-core algorithm. All nodes with less links are gradually removed from the network graph. (B) Simplified P. falciparum PPI network by k-core algorithm.

Figure 3

Gene ontology of 154 core proteins extracted from Core5 PPI network was analyzed based on (A) cellular location and (B) function. Membrane and cell surface in (A) indicate those of parasite. The total number indicated in pie graphs (A and B) exceeds 154 as many proteins have multiple cellular locations and functions.

Figure 4

Bulk scale expression and purification of PfMSP1₁₉ and PvMSP1₁₉ recombinant proteins. M, protein size marker; Lane 1, total cell lysate; Lane 2, unbound fraction (flow-through); Lane 3, eluted fraction with washing buffer containing 40 mM imidazole; Lane 4, eluted fraction with elution buffer containing 500 mM imidazole. Arrows indicate purified recombinant proteins.

Figure 5

Clinical evaluation of PfMSP1₁₉ and PvMSP1₁₉ antigens by ELISA. PfMSP1₁₉ (A), PvMSP1_19ΔC (B) and PvMSP1_19ΔN (C) antigens were coated on the well and reacted with Pf-positive (Pf Ab(+)), Pv-positive (Pv Ab(+)), and Plasmodium-negative clinical samples. The identical samples were used for PvMSP1_19∆C (B) and PvMSP1_19∆N (C) in the same order.

Figure 6

Clinical evaluation of PfMSP1₁₉- and PvMSP1₁₉-based rapid diagnostic tests (RDTs). (A) PfMSP1₁₉-based Ab RDT (B) Comparison of diagnostic performance between PvMSP1₁₉-based Ab RDT (Novel) and SD BioLine Malaria P.f/P.v test kit (Commercial). Absorbance in ELISA: high (nos. 1 and 3), medium (nos. 7 and 10) and low (nos. 5 and 6).