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 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

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 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

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

RNA lab-on-chip analytical sensitivity using CHIKV spiked samples.

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

Tropical diseases, stratified by protozoal, bacterial, and viral infections, including causative agents, and current endemic areas.

<p>Tropical diseases, stratified by protozoal, bacterial, and viral infections, including causative agents, and current endemic areas.</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

Viral RNA extraction and amplification strategy in the detection of CHIKV.

<p>RNA extracted (4 µL) from respective spikes of 1 to 10⁵ PFU/µL was subjected to RNA lab-on-chip amplification or qRT-PCR assay. (A) Summary profiles of CHIKV specific probes and viral load quantification for respective spiked concentrations (n = 2). Hybridization profiles of RNA lab-on-chip of extracted viral RNA from spiked samples of (B) 10 PFU/µL and (C) 50 PFU/µL are shown. CHIKV specific probes are marked in blue.</p

Schematic illustration of tropical pathogen detection workflow.

<p>Sample processing steps included the isolation of total DNA and viral RNA from clinical samples, followed by amplification of extracted nuclei acids on the lab-on-chip, and hybridization of amplicon to target-specific capture probes (represented in blue). Both the amplification and hybridization processes are performed on the lab-on-chip. The steps leading to tropical pathogen identification (as represented in red) comprised of washing and drying of the chip, subsequent reading of the microarray in the optical reader, and software analysis of the microarray image.</p