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

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

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

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

<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

Additional file 1 of Falciparum malaria from coastal Tanzania and Zanzibar remains highly connected despite effective control efforts on the archipelago

Additional file 1. Additional figures and tables

Distribution of quantitative PCR determined parasite densities.

<p>pan- <i>Pan plasmodium</i>, Geometric mean values are indicated by horizontal lines. Geometric mean values are for PCR + 1.8 p/μL (range 0.1–770), for PCR+/ pan HTP-LAMP + 2.5 p/μL (range 0.2–770), for PCR+/ pan-HTP-LAMP– 1.4 p/μL (range 0.1–7) and for Chelex pan-LAMP + 3.4 p/μL (range 0.1–770).</p

Characterization of HTP-LAMP versus PCR discordant samples for diagnosis of <i>Plasmodium</i> infection (N = 31).

<p>Characterization of HTP-LAMP versus PCR discordant samples for diagnosis of <i>Plasmodium</i> infection (N = 31).</p