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

Comparison of molecular tests for the diagnosis of malaria in Honduras

Abstract Background Honduras is a tropical country with more than 70% of its population living at risk of being infected with either Plasmodium vivax or Plasmodium falciparum. Laboratory diagnosis is a very important factor for adequate treatment and management of malaria. In Honduras, malaria is diagnosed by both, microscopy and rapid diagnostic tests and to date, no molecular methods have been implemented for routine diagnosis. However, since mixed infections, and asymptomatic and low-parasitaemic cases are difficult to detect by light microscopy alone, identifying appropriate molecular tools for diagnostic applications in Honduras deserves further study. The present study investigated the utility of different molecular tests for the diagnosis of malaria in Honduras. Methods A total of 138 blood samples collected as part of a clinical trial to assess the efficacy of chloroquine were used: 69 microscopically confirmed P. falciparum positive samples obtained on the day of enrolment and 69 follow-up samples obtained 28 days after chloroquine treatment and shown to be malaria negative by microscopy. Sensitivity and specificity of microscopy was compared to an 18 s ribosomal RNA gene-based nested PCR, two single-PCR reactions designed to detect Plasmodium falciparum infections, one single-PCR to detect Plasmodium vivax infections, and one multiplex one-step PCR reaction to detect both parasite species. Results Of the 69 microscopically positive P. falciparum samples, 68 were confirmed to be P. falciparum-positive by two of the molecular tests used. The one sample not detected as P. falciparum by any of the molecular tests was shown to be P. vivax-positive by a reference molecular test indicating a misdiagnosis by microscopy. The reference molecular test detected five cases of P. vivax/P. falciparum mixed infections, which were not recognized by microscopy as mixed infections. Only two of these mixed infections were recognized by a multiplex test while a P. vivax-specific polymerase chain reaction (PCR) detected three of them. In addition, one of the day 28 samples, previously determined to be malaria negative by microscopy, was shown to be P. vivax-positive by three of the molecular tests specific for this parasite. Conclusions Molecular tests are valuable tools for the confirmation of Plasmodium species and in detecting mixed infections in malaria endemic regions.</p

Left ventricular hypertrophy in acute stroke patients with known hypertension

Background: Despite effective treatments, hypertension remains uncontrolled in nearly half of the people with hypertension in the United States. Uncontrolled hypertension leads to end organ damage, such as left ventricular hypertrophy (LVH). To identify reasons for uncontrolled hypertension, we interviewed acute stroke patients with a history of hypertension and evaluated for LVH. Methods: Using a standardized questionnaire, we collected demographic, socioeconomic, and health-care data in 300 acute ischemic and hemorrhagic stroke patients in one hospital. We also collected relevant clinical data from medical records. We analyzed factors associated with echocardiographic LVH as a marker of uncontrolled hypertension in 190 acute stroke patients with a history of hypertension. Results: Overall, 46% (88/190) of patients had LVH. In univariate analysis, lower household income and self-reported poor adherence to hypertension treatment were significantly associated with increased risk of LVH. In multiple logit modeling, only poor adherence to hypertension treatment remained significantly associated with LVH, odds ratio 1.77 (95% CI: 1.01–3.11), p < 0.05. Conclusions: In acute stroke patients, poor adherence to hypertension treatment is a significant independent predictor of LVH. A clear reason for poor adherence to treatment is elusive in a large proportion of these patients in our study. Further research is needed to identify and develop strategies to combat the key factors responsible for poor adherence to hypertension treatment

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

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