Additional file 1: of A simple, high-throughput, colourimetric, field applicable loop-mediated isothermal amplification (HtLAMP) assay for malaria elimination

Features of asymptomatic Ghanaian children samples (n=25). Table outlines features of the 25 samples chosen from a set of samples from asymptomatic school children in Kumasi, Ghana

Haplotypes of <i>Pfcrt</i>, <i>Pfmdr1</i> and <i>Pfdhfr</i> genes of <i>P</i>. <i>falciparum</i> isolates from pregnant women.

<p><b><i>Pfcrt</i></b> (n = 54): <i>CVMNK</i> (wild), <i>CVIET</i> (mutant); <b><i>Pfmdr1</i></b> (n = 28): NYSND (wild), <b>Y</b>YSND (single mutant), N<b>F</b>SND (single mutant), <b>YF</b>SND (double mutant); <b><i>Pfdhfr</i></b> (n = 15): ACNCSVI (wild), AC<b>I</b>C<b>N</b>VI (double mutant), AC<b>IRN</b>VI (triple mutant).</p

The laboratory code numbers are inicated in each box in the respective histograms

<p><b>Copyright information:</b></p><p>Taken from "Establishment of the 1World Health Organization International Standard for DNA for nucleic acid amplification technique (NAT)-based assays"</p><p>http://www.malariajournal.com/content/7/1/139</p><p>Malaria Journal 2008;7():139-139.</p><p>Published online 24 Jul 2008</p><p>PMCID:PMC2518157.</p><p></p

Location of TH2 and TH3 epitopes in the Structure of Circumsporozoite Protein.

<p>The relative location of TH2 (blue) and TH3 (pink) are shown within the protein structure.</p

Shared TH2 between Malawi and the Gambia.

<p>Shared TH2 between Malawi and the Gambia.</p

WebLogo of Amino Acid Sequence of Circumsporozoite Protein from Malawi and the Gambia.

<p>Panel A and Panel B are the Weblogos for Malawi and the Gambia, respectively. In Panel A, the TH2 region (blue) and TH3 region (pink) are underlined. The TH2 epitope maps almost exclusively to the α-helix, while the TH3 epitope maps to the flap. The polymorphic residues and types of amino acids that populate these sites appear to be conserved between two geographically disparate African parasite populations. Bits represent the information content, which is a relative measurement of sequence conservation, with higher values representing conservation and lower values consistent with sequence diversity at a position.</p

Calculated ΔΔG of observed polymorphic amino mutations from the ancestral amino acid residue compared relative to median of all possible mutations at each position.

<p>Free energy changes of polymorphisms in TH2 and TH3 are shown relative to the median change from all 19 substitutions from the predicted ancestral allele determined from <i>Plasmodium sp.</i> phylogeny. Mutations that have higher energy than the median are shown in red, while those with lower energy are shown in blue. Positive values represent increases in free energy and thermodynamic instability while negative values represent decline in free energy and greater stability. Neutral sequence where energetics have no effect would be expected to occur 50/50 above and below the median, while conservation of intramolecular function would be expected to minimize entropy and lead to lower energy states. Intermolecular interactions can lead to selection for less favorable states which are significantly enriched in the observed polymorphisms (17 increased vs 5 decreased, p = 0.00845).</p

TH2 and TH3 Pairings from Malawi and the Gambia.

<p>(a) This figure shows all TH2-x/TH3-y haplotype pairings comparing observed and expected. Those that are that are statistically over and under represented based upon our contingency table analysis (p≤0.00009 for Malawi and p≤0.003 for the Gambia) are colored blue. (b) The data shown are for those pairings in Malawi (blue) and the Gambia (green) that are either observed >5 times in our data, or those predicted to occur >5 times based upon our contingency analysis. Each pairing is represented a unique symbol. Of note, three pairings (TH2-1/TH3-1, TH2-6/TH3-1, and TH2-3/TH3-2) were over represented in both populations. In both figures, the diagonal line represents if there were non-random association of pairings based on predicted and observed values. Points above the line represent pairing over represented in the population, while those represented below the line are those under represented in the population. A complete list of these significant pairings is provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062427#pone.0062427.s005" target="_blank">Table S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062427#pone.0062427.s006" target="_blank">S3</a>.</p

Shared TH3 epitopes between Malawi and the Gambia.

<p>Shared TH3 epitopes between Malawi and the Gambia.</p

Significantly energetically-constrained amino acid positions identified by MUMBO Analysis.

<p>The 5 amino acids identified by MUMBO as having constrained ΔΔG mutational profiles relative to other identical amino acids within the crystal structure are color coded: ASN340 (red), Gly341 (orange) Ile342 (yellow) Glu343 (green), and Ser332 (cyan), are shown with respect to the TH2 and TH3 domains’ surface area (colored as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062427#pone-0062427-g004" target="_blank">Figure 4</a>). These residues cluster behind the conserved hydrophobic pocket and were identified because there mutational profile differed on average by 2 standard deviations from all other identical residues within the crystal structure.</p