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

Summary

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.

Abstract

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 0–1%. 10 µL 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.

Introduction

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 poor^1,2,3,4. 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 f....

Protocol

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

Results

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 .......

Discussion

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.......

Materials

List of materials used in this article

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