Eliminating Malaria from Haiti. Reinventing DNA to Eradicate Endemic Parasites

Firebird Biomolecular Sciences, Llc, Alachua FL

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Abstract

Eliminating Malaria from Haiti. Reinventing DNA to Eradicate Endemic Parasites Firebird Biomolecular Sciences LLC University of Florida Steven A. Benner John B. Dame Abstract Eradication of Plasmodium falciparum, the parasite that causes the most malarial deaths, is the only way to finally treat the disease in any specific geographical region. Haiti is one such region, where a small population (11 million), geographical isolation, and special ecology make eradication a real possibility. Thus, the Gates Foundation has just committed $29.4 MM to the CDC Foundation to attempt this eradication. Key to eradication is an assay that identifies human carriers, asymptomatic individuals with active parasite infections. That assay must: (a) Require only a small sample of blood, perhaps 100 µL; (b) Detect very small numbers of living parasites, perhaps as few as 10 parasites per sample; (c) Be as simple to operate as a conventional malarial ?rapid diagnostics test? (RDT); and (d) cost less than $1.00 to run. These specifications are virtually impossible with classical molecular diagnostics. However, Firebird has developed many innovations over the past three years that make such specs possible, including (a) sample prep processes that remove biohazard, (b) whole nucleic acid capture without centrifugation, (c) isothermal nucleic acid amplification using self-avoiding molecular recognition systems (SAMRS) and artifi- cially expanded genetic information systems (AEGIS), and (d) AEGIS molecular beacons. Firebird has shown that these support assays that, for example, detect 22 mosquito-borne RNA viruses in one mosquito carcass. The work is guided by work in the Dame lab showing that P. falciparum ribosomal RNA, present at ~104 copies per parasite, can be detected in malaria-infected blood using RT-PCR. The PCR assay does not meet the cost specs necessary for a LRE, and is not as easy to run as an RDT, but it shows the sensitivity of an assay directed at falciparum rRNA, where detection is robust if a sample of blood contains just one organism. This project will combine these innovations to develop an assay that detects falciparum rRNA as easily as an RDT, but which much higher sensitivity. One strength of the approach is its use of realistic samples of live falciparum in real blood to do benchmarking. Costing less than $1.00, the assay will generate fluorescence if falciparum is present that can be read directly or transmitted by cell phone camera for remote confirmation. We will benchmark (i) a sample preparation work-flow that releases P. falciparum rRNA that is (ii) captured on a solid support, where (iii) captured rRNA is amplified isothermally using primers with SAMRS and AEGIS nucleotides, with (iv) amplicons detected by AEGIS beacons. Results will be compared to samples analyzed by RT-PCR. Metrics for success include an ability to detect 10 parasites in a sample. In Phase 2, the efficacy of the test will be validated in a small field trial with blood from individuals from in a malaria-endemic area of Haiti, again comparing with RT-PCR. This will, as well, support the epidemiological science behind carrier identifica- tion and eradication, as we still do not know the lowest level of parasitemia that can remain stably in an individual. The product should gain WHO and Gates Foundation support, and yield LRE tests for other agents.