Detection and species identification of malaria parasites by isothermal tHDA amplification directly from human blood without sample preparation.

We report the clinical and analytical performance of an isothermal thermophilic helicase-dependent amplification assay for blood Plasmodium parasite detection and species-level identification. The assay amplifies the 18S rRNA gene fragment of all Plasmodium species and uses a species-specific probe and a pan-malarial probe to definitively identify Plasmodium falciparum from other infectious Plasmodium species. Amplicon-probe hybridization products are detected with a disposable dipstick enclosed in a cassette. With a pan-malarial-positive and P. falciparum-negative result, an additional test is performed to detect if the pan-malarial-positive band was the result of the presence of Plasmodium vivax. The assay uses only 2 μL of human whole blood directly for a 50-μL amplification reaction, without any pre-amplification processing. The clinical performance of the assay was validated using 88 samples from New York patients suspected of malaria or babesiosis. The overall sensitivity of the assay was 96.6% (95% CI, 87.3% to 99.4%), and the specificity was 100% (95% CI, 85.4% to 100%), compared with gold standard microscopy and a laboratory-developed molecular assay, respectively. The analytical sensitivity was 50 copies of DNA per assay or 200 parasites per microliter of blood, and the assay can detect samples with parasitemia levels <1%. This novel molecular diagnostic assay requires minimal laboratory instrumentation and uses un-processed blood as input; it can be readily performed in the field.