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

Scenarios for Low Carbon and Low Water Electric Power Plant Operations: Implications for Upstream Water Use

Electric sector water use, in particular for thermoelectric operations, is a critical component of the water-energy nexus. On a life cycle basis per unit of electricity generated, operational (e.g., cooling system) water use is substantially higher than water demands for the fuel cycle (e.g., natural gas and coal) and power plant manufacturing (e.g., equipment and construction). However, could shifting toward low carbon and low water electric power operations create trade-offs across the electricity life cycle? We compare business-as-usual with scenarios of carbon reductions and water constraints using the MARKet ALlocation (MARKAL) energy system model. Our scenarios show that, for water withdrawals, the trade-offs are minimal: operational water use accounts for over 95% of life cycle withdrawals. For water consumption, however, this analysis identifies potential trade-offs under some scenarios. Nationally, water use for the fuel cycle and power plant manufacturing can reach up to 26% of the total life cycle consumption. In the western United States, nonoperational consumption can even exceed operational demands. In particular, water use for biomass feedstock irrigation and manufacturing/construction of solar power facilities could increase with high deployment. As the United States moves toward lower carbon electric power operations, consideration of shifting water demands can help avoid unintended consequences

Alarm Sensors: Evaluating the Effectiveness in Reducing Elderly Inpatient Falls

Patient falls, particularly those within the elderly population, are an ongoing issue with inpatient hospital stays. Bed and chair sensors are used to alert staff when patients are exiting in an effort to prevent falls. Since the 1940’s, inpatient falls have been the largest single occurring incident. Falls can lead to injury, which often translates into increased costs, longer lengths of stay, and poor patient outcomes. As a result, healthcare has taken aim to alleviate falls by implementing fall prevention programs which include nursing education, training, and technical support to increase the implementation of alarm sensors. The literature review consisted of randomized control trials, which were found through the use of medical databases such as, CINAHL full text, Medline, Cochrane Library, and ProQuest Nursing and Allied Health Source. After reviewing the data, all four studies indicated that alarm sensors alone do not decrease the number of falls for elderly patients. Further research needs to be completed to determine if other interventions, such as patient teaching and daily falls risk evaluation, combined with alarm systems would be effective in decreasing fall rates. Keywords: accidental falls, sensor systems, elderly, safety, injuries, inpatient, incidents, prevention, alarms, monitorin

Evidence of an environmental effect on survival in ALS

Amyotrophic lateral sclerosis (ALS, motor neuron disease) is a neurodegenerative disorder of motor neurons leading to paralysis and eventual death by respiratory failure. Median survival is 2-3 years. Susceptibility genes, environmental triggers and disease related prognostic factors have been established, but environmental effects on survival are yet to be investigated. We analysed survival in the South-East England ALS register (SEALS register). Kaplan-Meier and Cox regression analyses were used to investigate survival in London, coastal and rural areas according to postcode at diagnosis. Results showed that there were 933 cases of ALS identified in the catchment area during the study period (1994-January 2012). Cox regression demonstrated a highly significant model for survival with significant protective variables: coastal residency, riluzole use and younger age at onset. Significantly worse survival was associated with London residency, older age as well as definite and probable El Escorial classifications. In conclusion, these findings suggest the possibility of an environmental effect on survival in ALS

MOESM1 of Plasmodium cynomolgi infections in rhesus macaques display clinical and parasitological features pertinent to modelling vivax malaria pathology and relapse infections

Additional file 1.  Haematological and parasitological data of Plasmodium cynomolgi B strain infections in rhesus macaques for up to 100 days. The data used in this study are contained within this file with appropriate metadata

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

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

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

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

Specificity results of the r64 primer set when tested against human-infecting and simian-infecting malaria parasites.

<p>All species were tested at least twice for reproducibility.</p