Additional file 1: of SENSory re-learning of the UPPer limb after stroke (SENSUPP): study protocol for a pilot randomized controlled trial

SPIRIT checklist. (DOC 120 kb

PCR and species results for the 65 ‘final positive’ field samples.

<p>Each row represents one sample. Pan, <i>Plasmodium spp</i>.; F, <i>P</i>. <i>falciparum</i>; M, <i>P</i>. <i>malariae</i>; FM, <i>P</i>. <i>falciparum</i> and <i>P</i>. <i>malariae</i> mixed infection; +, positive; −, negative.</p

The cytb-qPCR standard curves derived from tenfold dilution series of <i>P</i>. <i>falciparum</i>, <i>P</i>. <i>vivax</i>, <i>P</i>. <i>malariae</i>, and <i>P</i>. <i>ovale</i> samples.

<p>The cytb-qPCR efficiency (E) and coefficient of determination (R²) of the standard curves were calculated for each species. The cytb qPCR was conducted in eight replicates (4 extractions × 2 cytb-qPCRs). Dashed lines represent 95% CI.</p

The cytb-qPCR products and RFLP assays for species determination.

<p>a) The 430 bp cytb-qPCR products for each species. b-e) Four RFLP assays using FspBI, AluI, HpyCH4V and Csp6I restriction enzymes for <i>Plasmodium</i> species determination. f) Schematic diagram showing the RFLP product lengths. a-e) Lane 1, GeneRuler low range DNA ladder (Thermo Fisher); F, <i>P</i>. <i>falciparum</i>; V, <i>P</i>. <i>vivax</i>; M, <i>P</i>. <i>malariae</i>; O, <i>P</i>.<i>ovale</i>; NC, negative control.</p

Parasite detection limits for each PCR method.

<p>Solid dots represent PCR positive and hollow dots represent PCR negative samples. Detection limits are shaded in grey.</p

<i>Plasmodium</i> species detected by three PCR methods compared with the ‘final species’.

<p>^a The ‘final species’ is the combined species results of the three PCRs.</p><p>^b Mixed means <i>P</i>. <i>falciparum</i> and <i>P</i>. <i>malariae</i> mixed infection.</p><p>^c The kappa values were calculated by comparing individual PCRs with the ‘final species’.</p><p><i>Plasmodium</i> species detected by three PCR methods compared with the ‘final species’.</p

Proportion of pan-<i>Plasmodium</i> positive samples detected by each PCR compared with the ‘final positive’ samples.

<p>^a Proportion of samples positive in the pan-<i>Plasmodium</i> screening of field samples (N = 2977).</p><p>^b The <i>p</i> value, kappa value, sensitivity and specificity were calculated by comparing individual PCRs with the ‘final positive’ samples defined as positivity in at least two of the five PCR methods.</p><p>Proportion of pan-<i>Plasmodium</i> positive samples detected by each PCR compared with the ‘final positive’ samples.</p

Area chart showing the sequence alignments of target genes, primers and probes.

<p>a) Five copies of the 18S rRNA gene in the <i>P</i>. <i>falciparum</i> genome. b) Five copies of the 18S rRNA gene in the <i>P</i>. <i>vivax</i> genome. c) Combined ten copies of the 18S rRNA gene in the <i>P</i>. <i>falciparum</i> and <i>P</i>. <i>vivax</i> genomes. d) The cytb genes of <i>P</i>. <i>falciparum</i>, <i>P</i>. <i>vivax</i>, <i>P</i>. <i>malariae</i>, and <i>P</i>. <i>ovale</i>. Identical sequences are shown in grey in the chart area. Nucleotide base pair position is shown on the X-axis and the proportion of sequences in consensus is shown on the Y-axis. Primer and probe positions of respective PCRs are indicated using white arrows along the dashed lines (1, 18S-nPCR pan-<i>Plasmodium</i> primers; 1’, 18S-nPCR species-specific primers; 2, 18S-qPCR-R pan-<i>Plasmodium</i> primers and probe; 2’, 18S-qPCR-R species-specific primers and probes; 3, 18S-qPCR-K; 4, cytb-nPCR; and 5, cytb-qPCR).</p

Fever patient samples with discordant results.

<p>*Determined by blood smear microscopy, RDT = rapid diagnostic test + positive, − negative.</p><p>Outcome summery of fever patient samples with discordant results between Pan and/or Pf-LAMP and nested PCR using ABI extracted DNA and after DNA re-extraction with Chelex.</p

Sensitivities, specificities, positive and negative predictive values and kappa analysis.

<p><i>P.f.</i>* = <i>P. falciparum</i> mono infections (n = 33), P.m.** = <i>P.malariae</i> mono infections (n = 13), P.f.*** <i>P. falciparum</i> mono and mixed.</p><p>infections (n = 41), CI = confidence interval, PPV = positive predictive value, NPV = negative predictive value.</p><p>Sensitivities, specificities, positive and negative predictive values and kappa analysis for detection of malaria DNA from fever patients and asymptomatic individuals with Pan and Pf-LAMP versus gold standard (real- time PCR corrected Cytochrome b nested PCR).</p