MOESM1 of Streamlined, PCR-based testing for pfhrp2- and pfhrp3-negative Plasmodium falciparum

Additional file 1: Figure S1. Pfhrp2 assay performance using serially diluted P. falciparum 3D7 strain DNA. Elongation temperatures were varied as listed below. All other reaction conditions are specified in Table 1. Figure S2. Pfhrp3 assay performance using serially diluted P. falciparum 3D7 strain DNA. Elongation temperatures were varied as listed below. All other reaction conditions are specified in Table 1. Figure S3. Representative agarose gel electrophoresis depicting unexpected spurious bands from Dd2 strain (pfhrp2-deleted) control DNA. PCR targeting pfhrp2 exon 1/2 (assay 1 outer) yielded a spurious ~ 300 bp band from serial dilutions of pfhrp2-deleted Dd2 strain control DNA at all three elongation temperatures. Figure S4. Representative agarose gel electrophoresis depicting unexpected spurious bands from HB3 strain (pfhrp3-deleted) control DNA. PCR targeting pfhrp3 exon 1/2 (assay 5) yielded spurious bands at ~ 300, 400, and 800 bp from serial dilutions of Dd2 strain control DNA using optimized elongation temperatures (Table 1). Figure S5. Pfhrp3 assay performance using serially diluted P. falciparum 3D7 strain DNA. Elongation temperatures were varied as listed below. All other reaction conditions are specified in Table 1. The sequence of pfhrp2 exon 1/2 (assay 1) PCR product aligns to pfhrp3, due to spurious PCR amplification of the Dd2 pfhrp3 gene. PCR was performed using 0.01 ng/μL of 3D7 (pfhrp2-positive) and Dd2 (pfhrp2-negative) control DNA, respectively (see Additional file 1: Figure S3), followed by Sanger sequencing of amplicons. Reference sequences from the consensus 3D7 (v3.0) genome for pfhrp2 and pfhrp3 are displayed on the top two rows (REF), from 5′→ 3′, with capital letters for coding regions and genetic coordinates in reference to the pfhrp2 gene. Identical bases are indicated by a period (.), missing bases by a dash (-), substitutions by the discordant base. PCR product sequence contigs are highlighted as follows: 3D7 control DNA (light gray); Dd2 control DNA (dark gray). Figure S6. The sequences of pfhrp3 exon 1/2 (assay 5) PCR product align to pfhrp2, due to spurious PCR amplification of the HB3 pfhrp2 gene. PCR was performed using 0.01 ng/μL of 3D7 (pfhrp3-positive) and HB3 (pfhrp3-negative) control DNA, respectively (see Additional file 1: Figure S4), followed by Sanger sequencing of amplicons. Reference sequences from the consensus 3D7 (v3.0) genome for pfhrp2 and pfhrp3 are displayed on the top two rows (REF), from 5′→ 3′, with capital letters for coding regions and genetic coordinates in reference to the pfhrp3 gene. Identical bases are indicated by a period (.), missing bases by a dash (-), substitutions by the discordant base. PCR product sequence contigs are highlighted as follows: 3D7 control DNA (medium gray) and HB3 control DNA 300 bp fragment (light gray), 400 bp fragment (medium gray), and 800 bp fragment (dark gray

MOESM2 of Streamlined, PCR-based testing for pfhrp2- and pfhrp3-negative Plasmodium falciparum

Additional file 2. FASTA file containing Sanger sequences of amplicons produced by pfhrp2/3 exon 1/2 assays when applied to Dd2 (pfhrp2-negative), HB3 (pfhrp3-negative), and 3D7 (pfhrp2/3-positive) DN

MOESM1 of Individual and household characteristics of persons with Plasmodium falciparum malaria in sites with varying endemicities in Kinshasa Province, Democratic Republic of the Congo

Additional file 1. Baseline survey

MOESM7 of A deep sequencing approach to estimate Plasmodium falciparum complexity of infection (COI) and explore apical membrane antigen 1 diversity

Additional file 7. Comparison of demographic factors between sample sequenced individually (n=79) and pooled population cluster samples (N=821) with analysable sequence reads

MOESM2 of Individual and household characteristics of persons with Plasmodium falciparum malaria in sites with varying endemicities in Kinshasa Province, Democratic Republic of the Congo

Additional file 2: Table S1. PCR Primers

MOESM4 of A deep sequencing approach to estimate Plasmodium falciparum complexity of infection (COI) and explore apical membrane antigen 1 diversity

Additional file 4. Six internal quality control samples were PCR amplified and deep sequenced in duplicate. Expected (first column) and actual (sequencing control samples 1-6, averaged across duplicates) haplotype percentages are similar. The average percent error between duplicates was 4.4% (range 0.4-13.6%)

MOESM2 of A deep sequencing approach to estimate Plasmodium falciparum complexity of infection (COI) and explore apical membrane antigen 1 diversity

Additional file 2. Pfama1 heminested primer sequences and Multiplex identifier (MID) sequences

MOESM2 of Plasmodium falciparum genetic variation of var2csa in the Democratic Republic of the Congo

Additional file 2. Neighbour-joining tree of samples from DRC, Benin and Senegal, produced in Figtree ( http://tree.bio.ed.ac.uk/software/figtree/ ). Edges are coloured from red to blue according to their bootstrap percentage (black edges are terminal and so have no bootstrap value). Dotted lines leading away from the tree are coloured to indicate the origin of the sample

Non-inferiority plots for primary outcomes and mean birth weight.

<p>The figures show two-sided 90%, 95% and 99% confidence intervals (largest to smallest vertical bars, respectively), equivalent to one-sided 95%, 97.5% and 99.5% confidence intervals. The dashed blue vertical line indicates the non-inferiority margin. ATP, according to protocol population (adjusted for site); adjusted, ATP population adjusted for site, gravidity, age group, gestational age, ITN use and socio-economic status; ITT, intention to treat, (adjusted for site). Numbers included in the analyses and numeric values of the estimates are provided in the supplement (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132247#pone.0132247.s012" target="_blank">S4 Table</a>).</p

Distribution of birth weight (A) and hemoglobin concentration (B) at fourth ANC visit by intervention group.

<p>Birth weight distributions were estimated using data from 2183 women in the IPTp-SP group and from 2208 women in the ISTp-AL group. Hemoglobin distributions utilise data from 1534 women in the IPTp-SP group and from 1600 women in the ISTp-AL group.</p