Limit of detection of LAMP reactions using the boil and spin DNA extraction method.

Representative amplifications experiments using serial dilutions of samples of known parasitaemia (quantified using the WHO International Standard as calibrator).Quantification was achieved by extracting DNA from a parasite dilution series made in whole blood using a Qiacube and DNA blood mini kit (Qiagen, De Hilden). Extracted DNA was then quantified using the Shokoples real time PCR [28] against a within run standard curve created from DNA extracted from the WHO international standard using the same methodology. Parasite count per microLitre was calculated given an initial parasite burden of 10% in the WHO standard and an assumed red cell count of 5 x106 red cells per microLitre. Mean time to turbidity in minutes as follows: 10,000 p/μL = 15.8; 1,000 p/μL = 15.9; 100 p/μL = 16.7; 10 p/μL = 18.2; 1 p/μL = 24.5.</p

The contents of high throughput LAMP set-up packed into a transportable hard-cover case.

The contents of high throughput LAMP set-up packed into a transportable hard-cover case.</p

Classical antimalarial killing rate profiles.

<p><i>P. falciparum</i> viability time-course profiles of four classical antimalarial drugs, illustrating the wide range of speed-of-action measured. Viable parasite are cleared in response to fast-acting (art and chq), but not slow-acting (pyri and ato) drugs after 72 hours of treatment.</p

Parasite viability time-course in response to various concentrations of drugs.

<p><b>A</b>. <i>P. falciparum</i> viability time-course profiles for atovaquone, pyrimethamine, and artemisinin at concentrations corresponding to 1×, 3×, 10×, and 100× their respective IC₅₀. Error bars are SEM of at least 4 independent experiments. <b>B</b>. Values represented in panel A. No PRR or 99.9% PCT could be calculated for the 1× IC₅₀ conditions.</p

Schematic representation of the <i>in vitro</i> PRR assay.

<p><b>A</b>. Intraerythrocytic <i>P. falciparum</i> cultured at 0.5% parasitemia and 2% hematocrit is treated with drugs. The medium is exchanged and the drug replenished every 24 hours. Aliquots corresponding to 10⁵ parasites are taken out at defined time points, washed, and free-drug parasites cultured with fresh erythrocytes under limiting serial dilution conditions (see Material and Methods). Parasite growth is subsequently monitored after 21 days and confirmed after 28 days, allowing to calculate the initial number of viable parasite in the aliquot. <b>B</b>. Parasite viability measurement allows in turn to determine the drug lag phase (i.e. time needed to reach the maximal rate of killing), PRR over one life cycle, and 99.9% PCT (i.e. the time needed to decrease the number of viable parasites by 3 –log units). The data presented in this panel are for illustration purpose only. Axe Y shows log (viable parasites +1) to allow representation of logarithms when counting of number of viable parasites is equal to zero.</p

Limit of detection of LAMP reactions using the high throughput sample processing system.

<p>Representative amplifications experiments using serial dilutions of samples of known parasitaemia (quantified using the WHO International Standard as calibrator). Mean time to turbidity in minutes as follows: 10,000 p/μL = 17.5; 1,000 p/μL = 18.5; 100 p/μL = 19.4; 10 p/μL = 21.3; 1 p/μL = 22.9.</p

Comparison of metabolic and viability assays.

<p><b>A</b>. <i>P. falciparum</i> radio-labeled hypoxanthine incorporations after 48 hours of drug treatment at concentrations corresponding to 10× IC₅₀ with artemisinin, atovaquone, and azithromycin, reported as percentages of untreated controls. Data are averages of 12 repetitions from two independent experiments <b>B</b>. <i>P. falciparum</i> viability after 48 hours of drug treatment at concentrations corresponding to 10× IC₅₀ with artemisinin, atovaquone, and azithromycin, reported as number of viable parasite, as determined by limiting serial dilutions. Data are averages of 4 independent experiments. In both panels, error bars represent the standard error of the mean (SEM).</p

Stratified analysis of parasitaemia comparing DBS and WB HTP-LAMP against nPCR with a cut-off at 2 p/μL.

<p>Stratified analysis of parasitaemia comparing DBS and WB HTP-LAMP against nPCR with a cut-off at 2 p/μL.</p

Parasite viability in response to various classical antimalarial drugs.

<p><b>A. </b><i>P. falciparum</i> viability time-course profiles for chloroquine (chq), mefloquine (mef), piperaquine (pip), artemisinin (art), lumefantrine (lum), pyronaridine (pyro), pyrimethamine (pyri), and atovaquone (ato). Error bars represent the SEM of at least 4 independent experiments. <b>B</b>. and <b>C</b>. Scatter plots of the compounds tested reporting the IC₅₀ versus the log(PRR) and 99.9% PCT, respectively. The dotted line in panel B is a log linear regression, the slope thereof is not significantly different from zero (<i>p</i> = 0.48). Data of panel C do not converge enough to establish a regression line.</p

The transportable hard-cover case containing the high throughput LAMP set-up.

<p>The transportable hard-cover case containing the high throughput LAMP set-up.</p