Name: detection and quantification of plasmodium falciparum in aqueous red blood cells by attenuated total reflection infrared spectroscopy and multivariate data analysis
Uploaded: 2018-11-02T13:30:00.000+00:00
Duration: 10 min 50 s
Description: Watch this Scientific Journal Video about Detection and Quantification of Plasmodium falciparum in Aqueous Red Blood Cells by Attenuated Total Reflection Infrared Spectroscopy and Multivariate Data An

Funding to the authors was provided by the Australian Research Council (Future Fellowship FT120100926 to BRW), National Health and Medical Research Council of Australia (Program grant and Senior Research Fellowship to JGB; Early Career Fellowships JSR; Infrastructure for Research Institutes Support Scheme Grant to the Burnet Institute), and the Victorian State Government Operational Infrastructure Support Grant to the Burnet Institute. We acknowledge Mr. Finlay Shanks for instrumental support.

Please consult appropriate Material Safety Data Sheets (MSDS) and seek appropriate Biosafety Level 2 (BSL-2) training. All culturing steps must be done in a BSL-2 cabinet using aseptic technique, meaning there is a risk of exposure to harmful UV radiation from decontamination steps, needle stick injuries, and potential biological exposure and infection if the parasite culture enters any injuries. Furthermore, stock blood from blood banks is only screened for certain diseases and the potential for spreading blood borne diseases is a potential risk. Seek immediate medical aid in the occurrence of injury.
1. Preparation and Measurement of 3D7 Plasmodium falciparum Parasite Dilution Series
NOTE: Plasmodium sp. culture is highly sensitive. Use fresh/unexpired reagents and feed the parasites regularly by changing the media. Feed cultures below 5% parasitemia every second day; feed cultures between 6-10% parasitemia once or twice a day; and feed cultures between 11&#8211;20% up to 4 times a day. Parasites that are beginning to starve will lose their shape and begin to contract. In such a case, feed and dilute immediately by changing the media and adding stock RBCs. Collect the donor blood in blood collection tubes containing heparin as the anticoagulant and measure within the first 6 h.
<ol>
	<li>Culture a total of 30 mL of 3D7 strain Plasmodium falciparum parasites according to the standard protocol until the parasitemia reaches 10% rings.</li>
	<li>Synchronization of parasites
	<ol>
		<li>11 h after shizogeny into the parasite lifecycle, resuspend the culture and transfer into a 50 mL conical tube using an automated pipette.</li>
		<li>Centrifuge the culture at 300&#8211;400 x g and standard laboratory conditions (25&#176;C and 100 kPa) for 5 min.</li>
		<li>Remove the supernatant by drawing it up with a pipette or using a Pasteur pipette attached to a vacuum without disturbing the pellet. Discard waste media into a 10% bleach solution.</li>
		<li>Slowly add 12&#8211;15 mL of 4% sorbitol solution to the pellet using an automated pipette and mix the culture by capping and inverting the tube until the culture is homogenous.</li>
		<li>Incubate the culture at 37 &#176;C for 15 min.</li>
		<li>Centrifuge the culture at 300&#8211;400 x g and standard laboratory conditions for 5 min.</li>
		<li>Remove the supernatant as described in step 1.2.3.</li>
		<li>Add 10&#8211;15 mL of 0.9% saline to the pellet and mix the solution by capping and inverting the tube until the culture is homogenous.</li>
		<li>Repeat the steps 1.2.6&#8211;1.2.8 twice more to wash all remnants of sorbitol.</li>
	</ol>
	</li>
	<li>Preparation of the parasitemia dilution series
	<ol>
		<li>Wash stock RBCs as in steps 1.2.6&#8211;1.2.9.</li>
		<li>Centrifuge the culture at standard laboratory conditions, stock RBCs at 300&#8211;400 x g for 5 min and discard supernatant as in step 1.2.3.</li>
		<li>Label microcentrifuge tubes as 0%, 0.010%, 0.025%, 0.075%, 0.100%, 0.250%, 0.750% and 1.000%, and add 0 &#181;L, 1 &#181;L, 2.5 &#181;L, 7.5 &#181;L, 10 &#181;L, 25 &#181;L, 75 &#181;L and 100 &#181;L of culture respectively using a 0.2-20 &#181;L pipette.</li>
		<li>Add 100 &#181;L, 99 &#181;L, 97.5 &#181;L, 92.5 &#181;L, 80 &#181;L, 75 &#181;L, 25 and 0 &#181;L of stock RBCs to the tubes labelled 0%, 0.010%, 0.025%, 0.075%, 0.100%, 0.250%, 0.750%, and 1.000%, respectively.</li>
		<li>Centrifuge the fresh donor blood collected in anticoagulant tubes at 1,200 x g and standard laboratory conditions for 10 min.</li>
		<li>Remove the plasma by drawing it up with a pipette and discarding as described in step 1.2.3.</li>
		<li>Add 900 &#181;L of isolated RBCs into each microcentrifuge tube and mix thoroughly by inverting 10x.</li>
	</ol>
	</li>
	<li>Spectral acquisition
	<ol>
		<li>Using the accompanying spectral acquisition program, click &#34;Instrument Set-Up&#34; and set the data collection parameters to the following: 128 scans for background; 32 scans for sample; and resolution to 8/cm over the maximum sample range of the instrument.</li>
		<li>Set the temperature of the ATR-FTIR spectrometer per manufacturer&#39;s recommendation or overnight.</li>
		<li>Clean the crystal by gently scrubbing in a circular motion using lint free wipes dampened with ultrapure water. Then thoroughly dry using another lint free wipe.</li>
		<li>Take a background measurement of the air by clicking &#34;Background Measurement&#34;. Every 20 min, clean the crystal as in step 1.4.3 and repeat this measurement.</li>
		<li>Open the live view by clicking &#34;Preview&#34;. Observe a flat, horizontal baseline that indicates that the crystal is clean.</li>
		<li>Pipette 10 &#181;L of deionized water directly on to the middle of the crystal and click &#34;Measure Sample&#34;. Repeat this water measurement after every fifth sample.</li>
		<li>Dry the crystal using a lint free wipe and a gentle circular motion.</li>
		<li>Pipette 10 &#181;L of sample and click &#34;Measure Sample&#34;.</li>
		<li>Clean the crystal between samples as in step 1.4.3.</li>
		<li>Repeat until 3 replicates are acquired, making sure to randomize the order of both the samples and the replicates.</li>
	</ol>
	</li>
</ol>
2. Multivariate Data Analysis (MVDA)
NOTE: Data treatment must be done over the whole dataset in order to avoid noise and the addition of peaks of non-biological origin. In contrast, analyze over the biologically relevant regions: 2980-2800/cm and 1750-850/cm.
<ol>
	<li>Data treatment 
	NOTE: Two software, e.g., Matlab (henceforth referred to as software 1) and The Unscrambler X (henceforth referred to as software 2) are used as examples for MVDA. Software 1 has the capability of performing MVDA without the addition of a graphical user interface (GUI). However, it is recommended to purchase a GUI (Table of Materials). The following instructions when referring to software 1 will assume that is in conjunction with the commercially available GUI toolbox, and the example data treatment script has been attached.
	<ol>
		<li>Open the appropriate multivariate data analysis software, click &#34;Import Data&#34; and select the type of file for analysis.
		<ol>
			<li>In software 1, input &#34;Analysis&#34; into the command window to open the GUI. Right click the X box to find &#34;Import Data&#34;.</li>
			<li>In software 2, click the tab &#34;File&#34; to find &#34;Import&#34;.</li>
		</ol>
		</li>
		<li>Import sample, water and baseline spectra as data sets into the workspace by selecting all spectra in each set separately and clicking &#34;Open&#34; and give each set a short name, e.g., &#39;wat&#39; for dataset of water spectra.</li>
		<li>Select new table/matrix by clicking &#34;New Matrix/Vector&#34; and generate a n&#215;1 vector, where n is the number of samples. Input the parasitemia of each sample and give the vector the name &#39;Parasitemia&#39;.
		<ol>
			<li>In software 1, click &#34;New Variable&#34; in the command window.</li>
			<li>In software 2, click the icon &#34;New Matrix&#34;.</li>
		</ol>
		</li>
		<li>Plot data by clicking the &#34;Plot Data Icon&#34;. Inspect the spectra for water vapor effects by clicking &#34;Zoom&#34; and zooming in on 1800-1400/cm; most clearly observed as short, sharp, narrow peaks along the slopes of the amide I and amide II bands.</li>
		<li>In cases of extreme water vapor, open edit/pre-process data tab, and select &#34;Smoothing&#34;. Reduce the noise and/or strong water vapor contributions by smoothing the sample and water spectra using up to 25 points of smoothing or use a water vapor correction method.</li>
		<li>Correct non-horizontal baseline by using the baseline correction algorithm if appropriate, under the same edit/pre-process data tab in step 2.1.4.</li>
		<li>Average water spectra and copy the rows into an n&#215;m matrix equivalent to the sample dataset and reduce it to 70% intensity by multiplying it by 0.7.
		<ol>
			<li>In software 1, do so in the command window by inputting the following script &#34;AverageWater=mean(WaterDataset)&#34;. Then copy-paste the rows to match the sample data set. To reduce the intensity, input &#34;AverageWater70= AverageWater*0.70&#34;.</li>
		</ol>
		</li>
		<li>Subtract average water spectra from each sample spectrum.
		<ol>
			<li>In software 1, do so in the command window by inputting the following script &#34;WaterCorrectedData=SampleDataset-AverageWater70&#34;.</li>
		</ol>
		</li>
		<li>Open the edit/pre-process data tab to apply a second derivative function, and then normalize the data by selecting single normal variate (SNV) function and mean center data.
		<ol>
			<li>In software 1, do so in one go. First select &#34;Derivative&#34;, and input 25 points of smoothing, polynomial order of 3 and derivative order on the sample set using 25 points of smoothing and a Savitzky-Golay function. Then select &#34;SNV&#34; and &#34;Mean Center&#34;. Click &#34;Okay/Apply&#34;.</li>
		</ol>
		</li>
		<li>Open the edit/pre-process data tab and in &#34;Column Variables&#34;, select 2,980&#8211;2,800/cm and 1,750&#8211;850/cm by making sure only their boxes are ticked.</li>
	</ol>
	</li>
	<li>Data analysis
	<ol>
		<li>Principal Component Analysis (PCA)
		<ol>
			<li>Click &#34;Analysis | Decomposition&#34; and select PCA.
			<ol>
				<li>In software 1, click &#34;Build Model&#34;.</li>
				<li>In software 2, input the PCA methods. Input the optimal number of Principal Components (PCs) -in this work, 7- and a maximum of 100 iterations, and select cross-validation for the validation method. Click &#34;Run&#34;.</li>
			</ol>
			</li>
			<li>Observe the 95% confidence limit, the dashed ring, on the scores plot between PC1 and PC2.</li>
			<li>Mark the sample spectra with scores that occur outside the limit as potential outliers using the &#34;Select Spectra Tool&#34;.</li>
			<li>If the marked spectra have high Hotellings T2 values and high Q residuals exclude them from further analysis.</li>
		</ol>
		</li>
		<li>Partial Least Squares Regression (PLS-R)
		<ol>
			<li>Click &#34;Analysis | Regression&#34; and select &#34;PLS-R&#34;.
			<ol>
				<li>In software 1, right click Y block and select the vector &#34;Parasitemia | Build Model&#34;.</li>
				<li>In software 2, input the PLS-R methods. Select the vector &#34;Parasitemia&#34; as the &#34;Y reference&#34; and the sample dataset as &#34;X Data Set&#34; and select cross-validation as the validation method. Click &#34;Run&#34;.</li>
			</ol>
			</li>
			<li>Analyze the regression model. R2 values over 0.80 and root mean square error of cross-validation (RMSECV) values that are less than 0.1% parasitemia are acceptable models.</li>
			<li>Analyze the regression vector and identify the biological bands.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Hommel, M., Gilles, H., Collier, L., Balows, A., Sussman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+20.">Chapter 20.</a> Topley &amp; Wilson's Microbiology and Microbial Infections. Vol. 5 Parasitology. , 361 (1998).</li><li>World Health Organization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> World Malaria Report 2016. , (2016).</li><li>Rogers, W., Murray, P., Baron, E., Pflaller, M., Tenover, F., Yolken, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+105.">Chapter 105.</a> Manual of Clinical Microbiology. , 1355 (1999).</li><li>White, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+9.">Chapter 9.</a> Manson's Tropical Infectious Diseases. 23, 532-600 (2014).</li><li>Lindblade, K. A., Steinhardt, L., Samuels, A., Kachur, S. P., Slutsker, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+silent+threat:+asymptomatic+parasitemia+and+malaria+transmission.">The silent threat: asymptomatic parasitemia and malaria transmission.</a> Expert Rev Anti Infect Ther. 11 (6), 623-639 (2013).</li><li>Hanscheid, T., Grobusch, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+useful+is+PCR+in+the+diagnosis+of+malaria.">How useful is PCR in the diagnosis of malaria.</a> Trends Parasitol. 18 (9), 395-398 (2002).</li><li>Joanny, F., L&#246;hr, S. J., Engleitner, T., Lell, B., Mordm&#252;ller, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limit+of+blank+and+limit+of+detection+of+Plasmodium+falciparum+thick+blood+smear+microscopy+in+a+routine+setting+in+Central+Africa.">Limit of blank and limit of detection of Plasmodium falciparum thick blood smear microscopy in a routine setting in Central Africa.</a> Malar J. 13 (1), 234-241 (2014).</li><li>World Health Organisation. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Malaria Rapid Diagnostic Test Performance: results of WHO product testing of malaria RDTs: round 6 (2014-2015). , (2014).</li><li>Khoshmanesh, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+quantification+of+early-stage+malaria+parasites+in+laboratory+infected+erythrocytes+by+attenuated+total+reflectance+infrared+spectroscopy+and+multivariate+analysis.">Detection and quantification of early-stage malaria parasites in laboratory infected erythrocytes by attenuated total reflectance infrared spectroscopy and multivariate analysis.</a> Anal Chem. 86, 4379-4386 (2014).</li><li>Martin, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+common+anticoagulants+in+detection+and+quantification+of+malaria+parasitemia+in+human+red+blood+cells+by+ATR-FTIR+spectroscopy.">The effect of common anticoagulants in detection and quantification of malaria parasitemia in human red blood cells by ATR-FTIR spectroscopy.</a> Anal. 142 (8), 1192-1199 (2017).</li><li>Fabian, H., M&#228;ntele, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of Vibrational Spectroscopy. , (2006).</li><li>Whelan, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+the+reversible+B+to+A-like+transition+of+DNA+in+eukaryotic+cells+using+Fourier+transform+infrared+spectroscoptMonitoring+the+reversible+B+to+A-like+transition+of+DNA+in+eukaryotic+cells+using+Fourier+transform+infrared+spectroscopy.">Monitoring the reversible B to A-like transition of DNA in eukaryotic cells using Fourier transform infrared spectroscoptMonitoring the reversible B to A-like transition of DNA in eukaryotic cells using Fourier transform infrared spectroscopy.</a> Nucleic Acid Res. 39 (13), 5439-5448 (2011).</li><li>Wood, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+importance+of+hydration+and+DNA+conformation+in+interpreting+infrared+spectra+of+cells+and+tissues.">The importance of hydration and DNA conformation in interpreting infrared spectra of cells and tissues.</a> Chem Soc Rev. 45, 1980-1998 (1980).</li><li>Plyler, E. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infrared+spectra+of+methanol,+ethanol,+and+rn-propanol.">Infrared spectra of methanol, ethanol, and rn-propanol.</a> J Res Natl Bur Stand. 48 (4), (1952).</li></ol>

The authors have nothing to disclose.

PLS-R model is a supervised multivariate method that finds a linear relationship, Y=bX+E, between the predictive variables X (here, the absorbance at each wavenumber) and a continuous variable Y (here, the parasitemia level). In short, the model combines the variables in X to create a new set of latent variables (LVs) that capture the variance on X correlated with Y, and computes a regression vector (b) that multiplied by a new spectrum results in the estimation of the y value. The strength of the model can be taken from...

Malaria is among the most devastating diseases of our time; over half the population lives at risk in endemic regions and it disproportionately burdens the poor1,2,3,4. A large part of the issue is the asymptomatic carriers and early stage patients that act as reservoirs for mosquito vectors5, causing spikes of infection during wet seasons and allowing it to persist in communities. Malaria is caused by five Plasmodium parasites, the most deadly of which is P. falciparum which causes the most severe form of the disease2.
Currently, techniques for the diagnosis of malaria are less than perfect. Optical microscopy, the current gold standard, can only detect 62-88 parasites/&#181;L depending on the method used6. Furthermore, due to the intensiveness and high skill required, in many regions microscopy misdiagnoses &#62;50% of cases, especially those with low parasitaemia levels2, which can be directly attributed to the lack of resources in the area and results in the misuse of anti-malarial drugs. The other 2 main diagnostic methods are Rapid Diagnostic Tests (RDTs), which utilize antibodies for the detection, and Polymerase Chain Reaction (PCR) assays, which discriminate and quantify parasites from DNA. Currently, RDTs are only able to detect P. falciparum and P. vivax of at least 100 parasites/&#181;L of blood resulting in rarer forms of the disease being untreated.7,8 In contrast, PCR assays discriminate and quantify different species of Plasmodium at a sensitivity of 0.0004-5 parasites/&#181;L of blood. However, it requires expensive reagents, equipment and technical skill, and thus is not suitable for the field application.
A highly sensitive, reliable and affordable technique is essential to improve diagnosis times, and thus improving patient outcomes, and making disease elimination possible. Attenuated Total Reflection Fourier transform (ATR-FTIR) spectroscopy offers a potential solution to this problem. Previous work has shown that it was possible to detect and quantify P. falciparum in methanol fixed blood films achieving the detection limits of &#60;1 parasite/&#181;L of blood (&#60;0.0002 % parasitemia)9, which is comparable to PCR methods. Recent studies have shown that it is possible to detect and quantify parasites in aqueous samples and thus eliminate the fixation step. However, factors such as water vapor, spectral noise and data treatment need to be taken into consideration for optimal results10.
This protocol aims to show new users how to acquire ATR-FTIR spectra and prepare a regression model for the detection of P. falciparum from aqueous Red Blood Cells (RBC) samples10.

<table><tbody><tr><td>Donor blood</td><td>-</td><td>-</td><td>Must be collected by a trained medical practitioner</td></tr><tr><td>Stock blood</td><td>Australian Red Cross`</td><td>-</td><td></td></tr><tr><td>3D7 Plasmodium falciparum</td><td>The Burnet Institute</td><td>-</td><td>Laboratory strain wild type equivalent</td></tr><tr><td>RPMI-1640 media with L-hepes without Sodium Bicarbonate</td><td>Sigma Aldrich</td><td>R6504</td><td></td></tr><tr><td>Albumax (Gibco)</td><td>Thermofisher</td><td>E003000PJ</td><td>Aliquot into 50 mL falcon tubes and store in freezer</td></tr><tr><td>Sodium Bicarbonate</td><td>Sigma Aldrich</td><td>S5761</td><td></td></tr><tr><td>Giemsa Stain</td><td>Sigma Aldrich</td><td>48900</td><td></td></tr><tr><td>Sorbitol</td><td>Sigma Aldrich</td><td>S1876</td><td>Must be filtered before use in culture</td></tr><tr><td>Blood collection tubes (Vacutainers)</td><td>Becton, Dickonson and Company</td><td>367671</td><td></td></tr><tr><td>Immersion Oil</td><td>Thermofisher</td><td>M3004</td><td></td></tr><tr><td>50 mL culture dishes</td><td>Falcon</td><td>353025</td><td></td></tr><tr><td>25mL pipette tips</td><td>Falcon</td><td>357515</td><td></td></tr><tr><td>10mL pipette tips</td><td>Falcon</td><td>357530</td><td></td></tr><tr><td>Microscope Slides</td><td>Sigma Aldrich</td><td>S8902</td><td></td></tr><tr><td>Centrifuge tubes 50 mL</td><td>Sigma Aldrich</td><td>T2318</td><td></td></tr><tr><td>Automated pipette controller</td><td>Integra-biosciences</td><td>155 015</td><td></td></tr><tr><td>Sorvall Legend X1R Centrifuge</td><td>Thermofisher</td><td>75004260</td><td></td></tr><tr><td>Forma&amp;trade; 310 Direct Heat CO2 Incubators</td><td>Thermofisher</td><td>310TS</td><td></td></tr><tr><td>Nikon Eclipse E-100 Binocular Microscope</td><td>Nikon Instruments</td><td>E100_2CE-MRTK-1</td><td>When purchasing ensure that the 100x lens is an oil immersion lens</td></tr><tr><td>Bruker Alpha Ft-IR Spectrometer with ATR Quick Snap Attachment</td><td>Bruker</td><td>9308-3700</td><td>Be sure to request &quot;Eco-ATR&quot; attachment when purchasing</td></tr><tr><td>Matlab</td><td>Mathworks Inc</td><td></td><td>Multivariate data analysis software</td></tr><tr><td>The Unscrambler X</td><td>CAMO</td><td></td><td>Multivariate data analysis software</td></tr><tr><td>PLS-Toolbox</td><td>Mathworks, Inc.</td><td></td><td>GUI for Matlab</td></tr></tbody></table>

detection and quantification of plasmodium falciparum in aqueous red blood cells by attenuated total reflection infrared spectroscopy and multivariate data analysis

We demonstrate a method of quantification and detection of parasites in aqueous red blood cells (RBCs) by using a simple benchtop Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) spectrometer in conjunction with Multivariate Data Analysis (MVDA). 3D7 P. falciparum were cultured to 10% parasitemia ring stage parasites and used to spike fresh donor isolated RBCs to create a dilution series between 0&#8211;1%. 10 &#181;L of each sample were placed onto the center of the ATR diamond window to acquire the spectrum. The sample data was treated to improve the signal to noise ratio and to remove the contribution of water, and then the second derivative was applied to resolve spectral features. The data were then analyzed using two types of MVDA: first Principal Component Analysis (PCA) to determine any outliers and then Partial Least Squares Regression (PLS-R) to build the quantification model.

Partial Least Squares (PLS-R) plot and its associated regression vector, Figure 1a and 1b respectively, show that the signal from parasites are distinct enough from the RBCs that they can be used to form a linear regression model to be used for the prediction of parasites in future data sets.
A robust, linear PLS-R model was generated with an R-squared value of 0.87 and a root mean ...

Watch this Scientific Journal Video about Detection and Quantification of Plasmodium falciparum in Aqueous Red Blood Cells by Attenuated Total Reflection Infrared Spectroscopy and Multivariate Data An

Detection and Quantification of Plasmodium falciparum in Aqueous Red Blood Cells by Attenuated Total Reflection Infrared Spectroscopy and Multivariate Data Analysis

Here, we present a protocol for the detection and quantification of Plasmodium falciparum in infected aqueous red blood cells using an attenuated total reflection infrared spectrometer and multivariate data analysis.

Detection and Quantification of Plasmodium falciparum in Aqueous Red Blood Cells by Attenuated Total Reflection Infrared Spectroscopy and Multivariate Data Analysis

We demonstrate a method of quantification and detection of parasites in aqueous red blood cells (RBCs) by using a simple benchtop Attenuated Total ...

detection-quantification-plasmodium-falciparum-aqueous-red-blood

Research

JoVE Journal

Chemistry

7.9K Views.  Monash University. Het algemene doel van deze procedure is het bouwen van een regressiemodel voor de voorspelling van malariaparasietmia met behulp van ATR-FTIR spectroscopie met multivariate data-analyse. Deze methode kan helpen versnellen point-of-care diagnostiek die kunnen helpen bij het verbeteren van de resultaten van patiënten in de toekomst. Het belangrijkste voordeel van deze techniek is dat, terwijl het model kan enige tijd duren om te bouwen, het is zeer draagbaar, gebruiksvriendelijk, zeer gevoelig...

Video: Detection and Quantification of Plasmodium falciparum in Aqueous Red Blood Cells by Attenuated Total Reflection Infrared Spectroscopy and Multivariate Data Analysis

Protocol for Plasmodium falciparum Infections in Mosquitoes and Infection Phenotype Determination

Once a gene is identified as potentially refractory for malaria, it must be evaluated for its role in preventing Plasmodium infections within the mosquito.    This protocol illustrates how the extent of plasmodium infections of mosquitoes can be assayed.  The techniques for preparing the gametocyte culture, membrane feeding mosquitoes human blood, and assaying viral titers in the mosquito midgut are demonstrated.

Once a gene is identified as potentially refractory for malaria, it must be evaluated for its role in preventing Plasmodium infections within the mosquito. This protocol illustrates how the extent of plasmodium infections of mosquitoes can be assayed. The techniques for preparing the gametocyte culture, membrane feeding mosquitoes human blood, and assaying viral titers in the mosquito midgut are demonstrated.

Once a gene is identified as potentially refractory for malaria, it must be evaluated for its role in preventing Plasmodium infections within the ...

Biology

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

P. falciparum causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully understood and several hypotheses have been put forward, including mechanical obstruction of microvessels by P. falciparum-parasitized red blood cells (pRBC). Indeed, during the intra-erythrocytic stage of its life cycle, P. falciparum has the unique ability to modify the surface of the infected erythrocyte by exporting surface antigens with varying adhesive properties onto the RBC membrane. This allows the sequestration of pRBC in multiple tissues and organs by adhesion to endothelial cells lining the microvasculature of post-capillary venules 1. By doing so, the mature forms of the parasite avoid splenic clearance of the deformed infected erythrocytes 2 and restrict their environment to a more favorable low oxygen pressure 3. As a consequence of this sequestration, it is only immature asexual parasites and gametocytes that can be detected in peripheral blood.
Cytoadherence and sequestration of mature pRBC to the numerous host receptors expressed on microvascular beds occurs in severe and uncomplicated disease. However, several lines of evidence suggest that only specific adhesive phenotypes are likely to be associated with severe pathological outcomes of malaria. One example of such specific host-parasite interactions has been demonstrated in vitro, where the ability of intercellular adhesion molecule-1 to support binding of pRBC with particular adhesive properties has been linked to development of cerebral malaria 4,5. The placenta has also been recognized as a site of preferential pRBC accumulation in malaria-infected pregnant women, with chondrotin sulphate A expressed on syncytiotrophoblasts that line the placental intervillous space as the main receptor 6. Rosetting of pRBC to uninfected erythrocytes via the complement receptor 1 (CD35)7,8 has also been associated with severe disease 9.
One of the most recently described P. falciparum cytoadherence phenotypes is the ability of the pRBC to form platelet-mediated clumps in vitro. The formation of such pRBC clumps requires CD36, a glycoprotein expressed on the surface of platelets. Another human receptor, gC1qR/HABP1/p32, expressed on diverse cell types including endothelial cells and platelets, has also been shown to facilitate pRBC adhesion on platelets to form clumps 10. Whether clumping occurs in vivo remains unclear, but it may account for the significant accumulation of platelets described in brain microvasculature of Malawian children who died from CM 11. In addition, the ability of clinical isolate cultures to clump in vitro was directly linked to the severity of disease in Malawian 12 and Mozambican patients 13, (although not in Malian 14).
With several aspects of the pRBC clumping phenotype poorly characterized, current studies on this subject have not followed a standardized procedure. This is an important issue because of the known high variability inherent in the assay 15. Here, we present a method for in vitro platelet-mediated clumping of P. falciparum with hopes that it will provide a platform for a consistent method for other groups and raise awareness of the limitations in investigating this phenotype in future studies. Being based in Malawi, we provide a protocol specifically designed for a limited resource setting, with the advantage that freshly collected clinical isolates can be examined for phenotype without need for cryopreservation.

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

This method investigates the platelet-mediated clumping phenotype of Plasmodium falciparum-infected erythrocytes (pRBC) in clinical isolates. This is performed by isolating and co-incubating platelet-rich plasma and a suspension of pRBC.

P. falciparum causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully ...

Immunology and Infection

Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means

Unlike other Plasmodium species, P. falciparum can be cultured in the lab, which facilitates its study 1. While the parasitemia achieved can reach the &asymp;40% limit, the investigator usually keeps the percentage at around 10%. In many cases it is necessary to isolate the parasite-containing red blood cells (RBCs) from the uninfected ones, to enrich the culture and proceed with a given experiment. 
When P. falciparum infects the erythrocyte, the parasite degrades and feeds from haemoglobin 2, 3. However, the parasite must deal with a very toxic iron-containing haem moiety 4, 5. The parasite eludes its toxicity by transforming the haem into an inert crystal polymer called haemozoin 6, 7. This iron-containing molecule is stored in its food vacuole and the metal in it has an oxidative state which differs from the one in haem 8. The ferric state of iron in the haemozoin confers on it a paramagnetic property absent in uninfected erythrocytes. As the invading parasite reaches maturity, the content of haemozoin also increases 9, which bestows even more paramagnetism on the latest stages of P. falciparum inside the erythrocyte.
Based on this paramagnetic property, the latest stages of P. falciparum infected-red blood cells can be separated by passing the culture through a column containing magnetic beads. These beads become magnetic when the columns containing them are placed on a magnet holder. Infected RBCs, due to their paramagnetism, will then be trapped inside the column, while the flow-through will contain, for the most part, uninfected erythrocytes and those containing early stages of the parasite.
Here, we describe the methodology to enrich the population of late stage parasites with magnetic columns, which maintains good parasite viability 10. After performing this procedure, the unattached culture can be returned to an incubator to allow the remaining parasites to continue growing.

Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means

The paramagnetic properties of hemozoin are used to isolate late stages of Plasmodium falciparum-infected red blood cells growing in culture. The method is simple and fast and does not affect the subsequent invasive capabilities of the parasites.

Unlike other Plasmodium species, P. falciparum can be cultured in the lab, which facilitates its study 1. While the parasitemia achieved can reach the ...

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of free merozoites from the blood by phagocytic cells. However assessing the functional efficacy of merozoite specific opsonizing antibodies is challenging due to the short half-life of merozoites and the variability of primary phagocytic cells. Described in detail herein is a method for generating viable merozoites using the E64 protease inhibitor, and an assay of merozoite opsonin-dependent phagocytosis using the pro-monocytic cell line THP-1. E64 prevents schizont rupture while allowing the development of merozoites which are released by filtration of treated schizonts. &#160;Ethidium bromide labelled merozoites are opsonized with human plasma samples and added to THP-1 cells. Phagocytosis is assessed by a standardized high throughput protocol. Viable merozoites are a valuable resource for assessing numerous aspects of P. falciparum biology, including assessment of immune function. Antibody levels measured by this assay are associated with clinical immunity to malaria in naturally exposed individuals. The assay may also be of use for assessing vaccine induced antibodies. &#160;

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Measuring antibody function is key to understanding immunity to Plasmodium falciparum malaria. This method describes the purification of viable merozoites, and measurement of opsonization-dependent phagocytosis by flow cytometry.

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of ...

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an exponential increase in the proportion of malaria infected RBCs, known as parasitemia. We describe a flow cytometry based protocol which utilizes a combination of the DNA dye Hoechst, and the mitochondrial membrane potential dye, JC-1, to identify RBCs which contain parasites and therefore the parasitemia, of in vivo blood samples from Plasmodium chabaudi adami DS infected mice. Using this approach, in combination with fluorescently conjugated antibodies, parasitized RBCs can be distinguished from leukocytes, RBC progenitors, and RBCs containing Howell-Jolly bodies (HJ-RBCs), with a limit of detection of 0.007% parasitemia. Additionally, we outline a method for the comparative assessment of merozoite invasion into two different RBC populations. In this assay RBCs, labeled with two distinct compounds identifiable by flow cytometry, are transfused into infected mice. The relative rate of invasion into the two populations can then be assessed by flow cytometry based on the proportion of parasitized RBCs in each population over time. This combined approach allows the accurate measurement of both parasitemia and merozoite invasion in an in vivo model of malaria infection.

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

The malaria parasite invades and replicates within red blood cells. The accurate assessment of merozoite invasion and parasitemia is therefore crucial in assessing the course of malaria infection. Here we describe a flow cytometry based protocol for the measurement of these parameters in a mouse model of malaria.

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an ...

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

The genetic variation responsible for the sickle cell allele (HbS) enables erythrocytes to resist infection by the malaria parasite, P. falciparum. The molecular basis of this resistance, which is known to be multifactorial, remains incompletely understood. Recent studies found that the differential expression of erythrocyte microRNAs, once translocated into malaria parasites, affect both gene regulation and parasite growth. These miRNAs were later shown to inhibit mRNA translation by forming a chimeric RNA transcript via 5&#39; RNA fusion with discreet subsets of parasite mRNAs. Here, the techniques that were used to study the functional role and putative mechanism underlying erythrocyte microRNAs on the gene regulation and translational potential of P. falciparum, including the transfection of modified synthetic microRNAs into host erythrocytes, will be detailed.&#160; Finally, a polysome gradient method is used to determine the extent of translation of these transcripts. Together, these techniques allowed us to demonstrate that the dysregulated levels of erythrocyte microRNAs contribute to cell-intrinsic malaria resistance of sickle erythrocytes.

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

Human microRNAs translocate from host erythrocytes to Plasmodium falciparum parasites. Here, the techniques used to transfect synthetic microRNAs into host erythrocytes and isolate all RNAs from P. falciparum are described. In addition, this paper will detail a method of polysome isolation in P. falciparum to determine the ribosomal occupancy and translational potential of parasite transcripts.

The genetic variation responsible for the sickle cell allele (HbS) enables erythrocytes to resist infection by the malaria parasite, P. falciparum. The ...

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes.
The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE1.
Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System2 (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types.

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes 1. The technique is adaptable and can be used on different types of genes, cells and organisms.

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by naturally acquired IgG antibodies specific for VAR2CSA. VAR2CSA is the parasite antigen that mediates the selective sequestration of IEs in the placenta that can cause a severe form of malaria in pregnant women, called placental malaria (PM). Protection from PM is mediated by VAR2CSA-specific antibodies that are believed to function by inhibiting placental sequestration and/or by opsonizing IEs for phagocytosis. The assay employs late-stage-synchronized IEs that have been selected in vitro to express VAR2CSA, plasma/serum-antibodies from women with naturally acquired PM-specific immunity, and the phagocytic cell line THP-1. However, the protocol can easily be modified to assay the functionality of antibodies to any parasite antigen present on the IE surface, whether induced by natural exposure or by vaccination. The assay offers simple and high-throughput evaluation, with good reproducibility, of an important functional aspect of antibody-mediated immunity in malaria. It is, therefore, useful when evaluating clinical immunity to P. falciparum malaria, a major cause of morbidity and mortality in the tropics, particularly in sub-Saharan Africa.

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The overall goal of this protocol is to provide instruction on how to measure the capacity of antibodies present in sera or plasma of individuals, naturally exposed to Plasmodium falciparum infection, to opsonize and induce phagocytosis of the parasite-infected erythrocytes (IEs).

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by ...

Standard Membrane Feeding Assay for the Detection of Plasmodium falciparum Infection in Anopheles Mosquito Vectors

Malaria remains one of the most devastating diseases worldwide and, to date, the African region is still responsible for 94% of all cases worldwide. This parasitic disease requires a protozoan parasite, an Anopheles mosquito vector, and a vertebrate host. The Anopheles genus comprises more than 500 species, of which 60 are known as vectors of the parasite. The Plasmodium parasite genus consists of 250 species, and 48 of these are involved in disease transmission. Furthermore, the Plasmodium falciparum parasite has contributed toward an estimated 99.7% of malaria cases in sub-Saharan Africa in recent years. 
Gametocytes form part of the sexual stage of the parasite and are ingested by the female mosquito upon feeding on an infected human host. Further development of the parasite within the mosquito is enhanced by favorable environmental conditions in the midgut of the mosquito. Here, the fusion of the female and male gametes takes place, and the motile ookinetes originate. The ookinetes enter the midgut epithelium of the mosquito, and mature ookinetes form oocysts, which, in turn, produce motile sporozoites. These sporozoites migrate to the mosquito's salivary glands and are injected as a mosquito takes a blood meal. 
For drug discovery purposes, mosquitoes were artificially infected with gametocyte-infected blood in the standard membrane feeding assay (SMFA). To detect infection within the mosquito and/or to assess the efficacy of antimalarial compounds, the midguts of the female mosquitoes were removed post infection and were stained with mercurochrome. This method was used to enhance the visual detection of oocysts under the microscope for the accurate determination of oocyst prevalence and intensity.

Standard Membrane Feeding Assay for the Detection of Plasmodium falciparum Infection in Anopheles Mosquito Vectors

The standard membrane feeding assay (SMFA) is regarded as the gold standard for the assessment and identification of potential antimalarial compounds. This artificial feeding system is used to infect mosquitoes to further evaluate the effects of such compounds on the intensity and prevalence of the Plasmodium falciparum parasite.

Malaria remains one of the most devastating diseases worldwide and, to date, the African region is still responsible for 94% of all cases worldwide. This ...

In Vitro Assay of Plasmodium-Infected Red Blood Cell Killing by Cytotoxic Lymphocytes

Malaria is a major public health concern, presenting more than 200 million cases per year worldwide. Despite years of scientific efforts, protective immunity to malaria is still poorly understood, mainly due to methodological limitations of long-term Plasmodium culture, especially for Plasmodium vivax. Most studies have focused on adaptive immunity protection against malaria by antibodies, which play a key role in controlling malaria. However, the sterile protection induced by attenuated Plasmodium sporozoites vaccines is related to cellular response, mainly to cytotoxic T lymphocytes, such as CD8+ and gamma delta T cells (&#947;&#948; T). Hence, new methodologies must be developed to better comprehend the functions of the cellular immune response and thus support future therapy and vaccine development. To find a new strategy to analyze this cell-mediated immunity to Plasmodium blood-stage infection, our group established an in vitro assay that measures infected red blood cell (iRBC) killing by cytotoxic lymphocytes. This assay can be used to study cellular immune response mechanisms against different Plasmodium spp. in the blood stage. Innate and adaptative cytotoxic immune cells can directly eliminate iRBCs and the intracellular parasite in an effector:target mechanism. Target iRBCs are labeled to evaluate cell viability, and cocultured with effector cells (CD8+ T, &#947;&#948; T, NK cells, etc.). The lysis percentage is calculated based on tested conditions, compared to a spontaneous lysis control in a flow cytometry-based assay. Ultimately, this killing assay methodology is a major advance in understanding cell-mediated immunity to blood-stage malaria, helping uncover new potential therapeutic targets and accelerate the development of malaria vaccines.

In Vitro Assay of Plasmodium-Infected Red Blood Cell Killing by Cytotoxic Lymphocytes

Here we describe a new method to help elucidate the mechanisms of cellular immunity to Plasmodium during the blood stage of infection. This is an in vitro assay that measures infected red blood cell killing by cytotoxic lymphocytes.&#160;

Malaria is a major public health concern, presenting more than 200 million cases per year worldwide. Despite years of scientific efforts, protective ...

Detection and Quantification of Plasmodium falciparum in Aqueous Red Blood Cells by Attenuated Total Reflection Infrared Spectroscopy and Multivariate Data Analysis

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