Name: 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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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 di...

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	<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. 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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&#8482; 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 &#34;Eco-ATR&#34; 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 ...

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