A New Single-Step PCR Assay for the Detection of the Zoonotic Malaria Parasite Plasmodium knowlesi

Recent studies in Southeast Asia have demonstrated substantial zoonotic transmission of Plasmodium knowlesi to humans. Microscopically, P. knowlesi exhibits several stage-dependent morphological similarities to P. malariae and P. falciparum. These similarities often lead to misdiagnosis of P. knowlesi as either P. malariae or P. falciparum and PCR-based molecular diagnostic tests are required to accurately detect P. knowlesi in humans. The most commonly used PCR test has been found to give false positive results, especially with a proportion of P. vivax isolates. To address the need for more sensitive and specific diagnostic tests for the accurate diagnosis of P. knowlesi, we report development of a new single-step PCR assay that uses novel genomic targets to accurately detect this infection.We have developed a bioinformatics approach to search the available malaria parasite genome database for the identification of suitable DNA sequences relevant for molecular diagnostic tests. Using this approach, we have identified multi-copy DNA sequences distributed in the P. knowlesi genome. We designed and tested several novel primers specific to new target sequences in a single-tube, non-nested PCR assay and identified one set of primers that accurately detects P. knowlesi. We show that this primer set has 100% specificity for the detection of P. knowlesi using three different strains (Nuri, H, and Hackeri), and one human case of malaria caused by P. knowlesi. This test did not show cross reactivity with any of the four human malaria parasite species including 11 different strains of P. vivax as well as 5 additional species of simian malaria parasites.The new PCR assay based on novel P. knowlesi genomic sequence targets was able to accurately detect P. knowlesi. Additional laboratory and field-based testing of this assay will be necessary to further validate its utility for clinical diagnosis of P. knowlesi

Molecular diagnosis of malaria by photo-induced electron transfer fluorogenic primers: PET-PCR.

There is a critical need for developing new malaria diagnostic tools that are sensitive, cost effective and capable of performing large scale diagnosis. The real-time PCR methods are particularly robust for large scale screening and they can be used in malaria control and elimination programs. We have designed novel self-quenching photo-induced electron transfer (PET) fluorogenic primers for the detection of P. falciparum and the Plasmodium genus by real-time PCR. A total of 119 samples consisting of different malaria species and mixed infections were used to test the utility of the novel PET-PCR primers in the diagnosis of clinical samples. The sensitivity and specificity were calculated using a nested PCR as the gold standard and the novel primer sets demonstrated 100% sensitivity and specificity. The limits of detection for P. falciparum was shown to be 3.2 parasites/µl using both Plasmodium genus and P. falciparum-specific primers and 5.8 parasites/µl for P. ovale, 3.5 parasites/µl for P. malariae and 5 parasites/µl for P. vivax using the genus specific primer set. Moreover, the reaction can be duplexed to detect both Plasmodium spp. and P. falciparum in a single reaction. The PET-PCR assay does not require internal probes or intercalating dyes which makes it convenient to use and less expensive than other real-time PCR diagnostic formats. Further validation of this technique in the field will help to assess its utility for large scale screening in malaria control and elimination programs

Quantitative comparison of PET-PCR assay and Rougemont real-time PCR assay.

<p>The PET-PCR and Rougemont real-time PCR assays were run using two well-quantified <i>P. falciparum</i> strains (Nigeria and Santa Lucia) used at six differing parasitemia levels (2000, 400, 80, 16, 3.2 and 0.64 parasites/µl). To determine the correlation between CT values and parasitemia, the mean CT values obtained in the PET-PCR assay (A) and in the Rougemont real time PCR assay (B) were plotted against the log transformed parasitemia (Log parasitemia). The CT values demonstrated reproducible linearity over the parasitemia range tested as both methods show significant correlation with R² values >0.990. No statistical difference in mean CT values was observed for the two different strains or for the two different methods (P>0.05).</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

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

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

Real-Time Loop-Mediated Isothermal Amplification (RealAmp) for the Species-Specific Identification of <em>Plasmodium vivax</em>

<div><p><em>Plasmodium vivax</em> infections remain a major source of malaria-related morbidity and mortality. Early and accurate diagnosis is an integral component of effective malaria control programs. Conventional molecular diagnostic methods provide accurate results but are often resource-intensive, expensive, have a long turnaround time and are beyond the capacity of most malaria-endemic countries. Our laboratory has recently developed a new platform called RealAmp, which combines loop-mediated isothermal amplification (LAMP) with a portable tube scanner real-time isothermal instrument for the rapid detection of malaria parasites. Here we describe new primers for the detection of <em>P. vivax</em> using the RealAmp method. Three pairs of amplification primers required for this method were derived from a conserved DNA sequence unique to the <em>P. vivax</em> genome. The amplification was carried out at 64°C using SYBR Green or SYTO-9 intercalating dyes for 90 minutes with the tube scanner set to collect fluorescence signals at 1-minute intervals. Clinical samples of <em>P. vivax</em> and other human-infecting malaria parasite species were used to determine the sensitivity and specificity of the primers by comparing with an 18S ribosomal RNA-based nested PCR as the gold standard. The new set of primers consistently detected laboratory-maintained isolates of <em>P. vivax</em> from different parts of the world. The primers detected <em>P. vivax</em> in the clinical samples with 94.59% sensitivity (95% CI: 87.48–98.26%) and 100% specificity (95% CI: 90.40–100%) compared to the gold standard nested-PCR method. The new primers also proved to be more sensitive than the published species-specific primers specifically developed for the LAMP method in detecting <em>P. vivax</em>.</p> </div

<i>P. vivax</i> r64 RealAmp primer set sequence (5′→3′).

<p><i>P. vivax</i> r64 RealAmp primer set sequence (5′→3′).</p