Development and analytical evaluation of a new Loop-mediated isothermal amplification assay for the specific detection of Plasmodium vivax infections

Background Malaria elimination needs to address the large number of infections harboring submicroscopic parasitaemia. The detection of these often asymptomatic infections requires more sensitive diagnostic tools than those commonly used for clinical case management such as light microscopy and rapid diagnostic tests. We report here the development and analytical evaluation of a new diagnostic kit for the detection of P. vivax based on loop-mediated isothermal amplification technology and temperature-stable dried-down reagents. Materials and Methods Primer design and production of dried-down reagents and prototype kits were conducted by Eiken Chemical Co. The laboratory evaluation of prototype kits included analytical specificity and sensitivity using a range of P. vivax, P. falciparum, P. ovale, and P. malariae isolates, P. knowlesi culture samples as well as negative whole blood samples. Results This new P. vivax LAMP kit is amenable to crude DNA extraction and all tests were conducted with DNA extracted using a simple “boil and spin” protocol. The analytical sensitivity was determined using 2-fold serial dilutions of two P. vivax infected blood samples of known parasitemia, ranging from 86,000 parasite/µL to 0.1 p/µL, and found to be 1 p/µL (3/3 and 2/3 repeats positive, respectively). The analytical specificity evaluation did not reveal any cross-species reactivity with P. falciparum at 19,000 p/µL, P. ovale samples at 10,000 p/µL, P. malariae samples at 20 p/µL and P. knowlesi samples at 350,000 p/µL. Conclusions This new prototype P. vivax LAMP assay shows excellent species-specificity and a limit of detection comparable to already existing Pan and P. falciparum specific LAMP kits. This new test complements existing LAMP kits and will facilitate population screening for the identification and removal of the asymptomatic reservoirs in P. vivax elimination campaigns. Field studies are currently ongoing to validate these encouraging laboratory results.