Rapid point-of-care diagnostic for malaria

Abstract

This work was supported by NIH funds UO1 Grant #DE017855 (DM). DM is a NYSTAR awardee and SM is a Gates Grand Challenge awardee. We gratefully acknowledge Drs. Karen Day and Jane Carlton (New York University) for reagents and OptiGene® for their technical collaboration. An alternative approach to a vaccine to alleviate the burden of malaria involves the “Test and Treat” paradigm currently being evaluated for eradication of HIV [1]. This approach predicts that if all symptomatic and asymptomatic individuals in a region are tested, and all of those who test positive are immediately treated, the burden of disease in that area will decrease. We believe that this approach is feasible for malaria with an inexpensive point-of-care diagnostic test for Plasmodium DNA, followed by appropriate therapeutic intervention. Currently, malaria is detected by (1) microscopic examination of thick and thin blood smears, a process that is both laborious and lengthy and requiring trained personnel; (2) laboratory-based PCR tests, which are expensive, time-consuming, and require specialized equipment and trained personnel; or (3) rapid antigen-based tests, which are generally insensitive. Commercial rapid-diagnostic tests (RDT) for malarial antigens were first introduced in 1994, and currently there are many such products available. The WHO has been evaluating these tests since 2009, and the third report assessing available RDTs was published in December 2012 [2]. The shortcomings with the available RDTs include: (1) Poor quality of some products; (2) Inability to detect mutant forms of Plasmodia that have deleted the histidine-rich protein 2 (HRP2) target gene; (3) Low sensitivity (500-1,000 parasites/μl); and (4) False positive reactions after the infection is cleared due to residual antigen in the bloodstream Isothermal amplification methods make use of polymerases with strand displacement capabilities that do not require a high temperature denaturation step. The entire amplification reaction occurs under isothermal conditions (65 C). The reaction is extremely efficient and yields an enormous amount of amplified DNA. Reaction progress is typically visualized using an intercalating dye. Our group designed a Point-of-Care (POC) Loop-mediated Isothermal Amplification (LAMP) nucleic acid detection test using either blood or oral fluid that has a sensitivity 10-100 fold greater than conventional PCR (6,7), 10-25 times more sensitive than antigen detection, requires no sample purification, and takes less than 30 min. Traditional LAMP utilizes two or three sets of primers that target the conserved 18S rRNA gene, however, we use primers that target the mitochondrial cytochrome C oxidase gene, which is 100 times more sensitive than other isothermal amplification methods currently available for Plasmodium falciparum (8) and is unlikely to be mutated or deleted.