Specimens included in this study.

*<p> = these included three mothers in investigations of possible congenital transmission and four organ donors with chronic Chagas disease. PCR-testing was performed on these chronically infected persons to aid in the evaluation of disease transmission risk.</p

Effect of storage of EDTA blood specimens on real-time PCR results.

<p>The lower the Cq (quantitative cycle) value, the better recovery was obtained from the DNA extraction process.</p

Detailed PCR findings from patients with reactivation disease.

<p>All of the patients in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001689#pntd-0001689-t003" target="_blank">Table 3</a> tested positive in serology for Chagas disease and no decrease in antibody titer was detected during the monitoring period.</p><p>BC = buffy coat; only the buffy coat fraction was PCR positive for these samples.</p>†<p>indicate specimens that were collected during drug treatment (benznidazole or nifurtimox). Drug treatment information is missing for patients #4 and #9.</p

Specimens included in this study.

*<p> = these included three mothers in investigations of possible congenital transmission and four organ donors with chronic Chagas disease. PCR-testing was performed on these chronically infected persons to aid in the evaluation of disease transmission risk.</p

Validation data for the real-time PCR assays.

<p>Validation data for the real-time PCR assays.</p

Detailed PCR findings for patients with acute Chagas disease.

1<p>Patient was serologically positive for Chagas disease by the time she was tested at CDC.</p>2<p>Patient was serologically positive when 19 days old due to maternal antibodies. Another sample collected at 10 months of age tested negative in serology.</p>†<p>indicate specimens that were collected during drug treatment (benznidazole or nifurtimox).</p

Detailed PCR-findings for recipients of organs from donors with suspected/confirmed chronic Chagas disease.

<p>All of the patients in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001689#pntd-0001689-t004" target="_blank">Table 4</a> were serologically negative at time of transplant and none sero-converted during the time they were monitored at CDC.</p><p>BC = buffy coat; only the buffy coat fraction was PCR positive for these samples.</p>†<p>indicate specimens that were collected during drug treatment (benznidazole or nifurtimox). Drug treatment information is missing for patients #4 and #9.</p

Limits of detection of primer set Pkr140-5.

<p>The analytical sensitivity of primer set Pk140-5 (A) and the primers from a published study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031848#pone.0031848-Singh1" target="_blank">[3]</a> (B) were determined using a well-quantitated <i>P. knowlesi</i> DNA standard. The blood sample was serially diluted ten-fold with a starting parasitemia of 100,000p/µl to 1p/µl. The expected base pair sizes for the two primers are included. Three different experiments are shown.</p

Specificity of the <i>P. knowlesi</i> primers tested using simian-infecting malaria species.

<p>To test the specificity of the <i>P. knowlesi</i> primers, 5 different simian-infecting malaria parasite species (<i>P. simiovale</i>, <i>P. inui</i>, <i>P. cynomolgi</i>, <i>P. hylobati and P. coatneyi</i>) including 3 different <i>P. knowlesi</i> isolates were tested. The no template control (NTC) was included as a negative control. A; primer set Pkr140-3 (expected size = 230bp), B; primer set Pkr140-4 (expected size = 280bp) and C; primer set Pkr140-5 (expected size = 200bp). Circles indicate non-specific amplification.</p

Spatial distribution of Pkr140 sequence targets across the 14 <i>P. knowlesi</i> genome.

<p>The circle represents chromosomes. Each chromosome is labeled with the 2-letter genus and species abbreviation for <i>P. knowlesi</i> and the chromosome number. Tick marks indicate 1 mb of sequence. Lines inside the circle indicate the location of Pkr140 copies and are not to scale. Circos 0.51 (<a href="http://mkweb.bcgsc.ca/circos/" target="_blank">http://mkweb.bcgsc.ca/circos/</a>) was used to generate this map <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031848#pone.0031848-Krzywinski1" target="_blank">[44]</a>.</p