Highly Sensitive and Rapid Quantitative Detection of Plasmodium falciparum Using an Image Cytometer.

Muneaki Hashimoto,Kazuaki Kajimoto,Hiroaki Oka,Musashi Matsumoto,Atsuro Tatsumi,Yoshihiro Nakajima,Noboru Minakawa,Kazumichi Yokota,Toshihiro Mita,Masatoshi Kataoka

doi:10.3390/microorganisms8111769

Abstract

The gold standard for malaria diagnosis is microscopic examination of blood films by expert microscopists. It is important to detect submicroscopic and asymptomatic Plasmodium infections in people, therefore the development of highly sensitive devices for diagnosing malaria is required. In the present study, we investigated whether an imaging cytometer was useful for the highly sensitive quantitative detection of parasites. Whole blood samples were prepared from uninfected individuals spiked with Plasmodium falciparum-infected erythrocytes. Thereafter, erythrocytes were purified using a push column comprising of a syringe filter unit with SiO2-nanofiber filters. After adding the erythrocytes, stained with nuclear stain, to a six-well plate, quantitative detection of the parasites was performed using an image cytometer, CQ1. Imaging of 2.6 × 106 erythrocytes was completed in 3 min, and the limit of detection indicated parasitemia of 0.00010% (≈5 parasites/μL of blood). In addition to rapid, highly sensitive, and quantitative detection, the ease of application and economic costs, image cytometry could be efficiently applied to diagnose submicroscopic parasites in infected people from endemic countries.

Highlights

According to the World Health Organization (WHO), in 2018, Plasmodium falciparum accounted for 99.7% of estimated malaria cases in Africa, the most endemic area of malaria
Because quantitative PCR is highly sensitive, it is often performed to detect parasites in the submicroscopic carriers [7]; the cost of qPCR is extremely high as it requires an expensive thermal cycling, high-speed centrifuge for the purification of DNA from blood samples, and a clean bench to avoid contamination
The filtered blood sample was diluted 100-fold with phosphate-buffered saline (PBS), and 10 μL of the aliquot was applied to a photon slide (Logos Biosystems, Inc., Gyeonggi-do, Korea)

Summary

Introduction

According to the World Health Organization (WHO), in 2018, Plasmodium falciparum accounted for 99.7% of estimated malaria cases in Africa, the most endemic area of malaria. Because endemic areas lack fully trained microscopists, rapid diagnosis tests (RDTs) have gained popularity. Recent reports have revealed that numerous parasite carriers maintain low levels of parasitemia, thereby increasing the need for highly sensitive diagnostic devices [4,5]. Because quantitative PCR (qPCR) is highly sensitive, it is often performed to detect parasites in the submicroscopic carriers [7]; the cost of qPCR is extremely high as it requires an expensive thermal cycling, high-speed centrifuge for the purification of DNA from blood samples, and a clean bench to avoid contamination. We developed diagnostic devices that quantitatively detect parasites marked with nuclear stain following erythrocyte purification with SiO2-nanofiber (NF) filters and forming monolayers of numerous erythrocytes on plastic substrates [8,9,10]. Malaria diagnosis using image cytometry has the potential to replace the present qualitative POCT using RDT, hopefully leading to the elimination of malaria

Parasite Culture

Preparation of Push Columns

Detection of Parasite-Infected Erythrocytes Using CQ1

Results and Discussion