Detection and Species Identification of Malaria Parasites by Isothermal tHDA Amplification Directly from Human Blood without Sample Preparation

Abstract

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.

A Field-Tailored Reverse Transcription Loop-Mediated Isothermal Assay for High Sensitivity Detection of Plasmodium falciparum Infections

“…This allowed processing of patients samples in just two minutes, followed by less than forty minutes reaction time to record a positive signal. Indeed, this study is the first report of a LAMP-based method, targeting parasite RNA instead of DNA in blood lysates as reported previously [66, 69]. A limitation of the whole blood lysis method presented in this study is in the large volume of lysis buffer needed to achieve an appropriate dilution (1:50) of the blood components, which appear to exhibit fluorescence quenching effects on the nucleic acid dyes used.…”

Emerging Technologies for the Clinical Microbiology Laboratory

“…Like LAMP, the isothermal amplification can be carried out using simple instrumentation in the absence of electricity (54). HDA has been applied to identification of C. difficile, Plasmodium spp., and S. aureus (55,56). An advantage of HDA is that detection of target can be achieved by incorporation of fluorescein or digoxigenin into the amplicon, followed by capture and visualization of the amplicon as a colored line on an enzyme immunoassay (EIA) lateral-flow strip (56)(57)(58).…”

“…HDA has been applied to identification of C. difficile, Plasmodium spp., and S. aureus (55,56). An advantage of HDA is that detection of target can be achieved by incorporation of fluorescein or digoxigenin into the amplicon, followed by capture and visualization of the amplicon as a colored line on an enzyme immunoassay (EIA) lateral-flow strip (56)(57)(58). This maintains the ability to utilize these assays without sophisticated instrumentation but also allows the detection of multiple targets in a single reaction.…”

Novel techniques and future directions in molecular diagnosis of malaria in resource-limited settings

“…Some of the targets currently detected in LAMP assays include the 18SrRNA gene, which is both pan-species and species-specific; the mitochondria, which detects both Plasmodium genus and P. falciparum; and the apicoplast genome which detects Plasmodium genus [23,25,26]. As with PCR, isothermal techniques have also been modified to identify different blood stages of the parasite [27] Other isothermal amplification techniques such as nucleic acid sequencebased amplification (NASBA), thermophilic helicase dependent amplification (tHDA) and recombinase polymerase amplification (RPA) have also been explored for malaria parasite detection, though they have not been used in endemic settings [28][29][30]. NASBA is particularly suited for RNA targets in a double-stranded DNA background with the ability of detecting gametocyte densities as low as 0.02-0.1/mL [31].…”