Antibody reactivity induced by immunization with irradiated sporozoites.

<p>Pooled serum samples from groups of mice immunized with sporozoites from the different parasite lines were analyzed by ELISA to assay IgG and IgM responses induced against <i>P. berghei</i> CSP (A), or <i>P. falciparum</i> CSP (B), using long peptides covering the N-terminus or the C-terminus of the antigen, and short peptides that include some of the repeat units of the central repetitive region. Serums were also tested by IFAT against wet sporozoites to detect anti-CSP IgG and IgM antibodies(C). Titres are expressed as the log of the highest dilution of serum giving a positive staining.</p

Sterile protection in mice immunized with <i>P. berghei</i> irradiated sporozoites and challenged with <i>P. berghei</i> or <i>P. berghei</i> [<i>PfCS</i>] sporozoites.

<p>Mice were immunized with 1 or 3 injections of <i>P. berghei</i> (indicated on the left of the panel) before challenge with 5 000 <i>P. berghei</i> or <i>P. berghei</i> [<i>PfCS</i>] sporozoites. All naive control mice developed a patent blood-stage infection. The data are representative of those obtained in duplicate experiments.</p

Sterile protection in [BALB/c×C57BL/6] F1 mice immunized three times with <i>P. berghei</i> or <i>P. berghei</i> [<i>PfCS</i>] and challenged with 5000 <i>P. berghei</i> or <i>P. berghei</i> [<i>PfCS</i>] sporozoites.

<p>All animal groups (5 mice per group) were monitored for blood-stage infections by examination of Giemsa-stained blood smears obtained daily from day 2 to day 11 post-challenge. All naive control mice developed a patent blood-stage infection.</p

Comparison of the amino acid sequences of the <i>P. berghei</i> ANKA clone cy17 (34) and <i>P. falciparum</i> Welcome strain (35) CSP polypeptides.

<p>Black dots represent identical amino acid residues while bars represent amino acid residues with similar characteristics. The repeat regions (including the pre- and post-repeat sequences) are underlined.</p

DNA extraction and amplification strategy in the detection of <i>P. falciparum</i>.

<p>DNA extracted (4 µL) from respective spike tests of 1 to 10³ parasites/µL was subjected to DNA lab-on-chip amplification or nested PCR assay. (A) Summary of <i>Plasmodium</i> genus-specific and <i>P. falciparum</i> specific positive probes for respective spiked concentrations (n = 2). The x-axis showed the different spiked concentrations used. The y-axis represented the number of positive probes at each dilution tier. Genus-specific probes are represented in red, while species-specific probes are in blue. Hybridization profiles of DNA lab-on-chip of extracted DNA from (B) 50 parasites/µL and (C) 100 parasites/µL spiked samples are shown. <i>Plasmodium</i> genus-specific probes are marked in red, while <i>P. falciparum</i> specific probes are marked in blue. Nested PCR can detect <i>P. falciparum</i> in spiked samples as low as 5 parasites/µl. The PCR products from the nested PCR assay were run on a 2% agarose gel electrophoresis. (D) Lane 1 = 0, lane 2 = 1, lane 3 = 5, lane 4 = 10, lane 5 = 50, lane 6 = 100, lane 7 = 500 and lane 8 = 1000 parasites/µL spiked samples. L: PCR Sizer 100 base pair DNA Ladder.</p

Association of microscopy and RT-PCR detection with lab-on-chip outcome.

<p>The outcome of the tropical pathogen chip test on clinical samples previously confirmed by microscopy or RT-PCR. (A) <i>P. falciparum</i>. (B). <i>P. vivax</i>. (C) CHIKV. (D). DENV 3. Histograms show the percentage of samples tested positive for <i>P. falciparum</i> (n = 64), <i>P. vivax</i> (n = 21), CHIKV (n = 27), and DENV 3 (n = 17) by DNA or RNA lab-on-chip. Statistical significance was measured using 2-sided Fisher exact test between the number of samples tested positive or negative for the respective pathogens by the chip on previously laboratory-confirmed samples (by reference methods). ****P<.0001.</p

Clinical performance of DNA chip on <i>P. vivax</i>.

<p>DNA lab-on-chip results for 23 <i>P. vivax</i> clinical isolates out of 170 specimens were compared with results from microscopy.</p

Flowchart detailing the screening and order of diagnostic testing of 160 samples received in Singapore and Thailand.

<p>Specimens positive for Plasmodium parasites were tested with lab-on-chip to evaluate the performance of the assay. Non-malaria samples were evaluated for CHIKV and DENV and subsequently tested with lab-on-chip assay for diagnostic methodology evaluation.</p

Clinical performance of RNA chip on CHIKV.

<p>RNA lab-on-chip results for 30 CHIKV clinical isolates out of 170 specimens were compared with results from qRT-PCR.</p

DNA lab-on-chip analytical sensitivity using <i>Plasmodium</i> spiked samples.

a<p>shows the number <i>Plasmodium</i> genus probes (out of two) with a fluorescence signal.</p>b<p>shows the number <i>P. falciparum</i> specific probes (out of two) with a fluorescence signal.</p>c<p>A positive detection for <i>P. falciparum</i> would require the presence of at least one of two probes (for both genus and <i>P. falciparum</i> specific) to give a positive fluorescence signal.</p>d<p>ND Not detected.</p