Development of a Multiplex Loop-Mediated Isothermal Amplification Assay for Diagnosis of Plasmodium spp., Plasmodium falciparum and Plasmodium vivax.

Woong Sik Jang,Hyunseul Jee,Younglan Choe,Chae Seung Lim,Minkyeong Choi,In Su Park,Chaewon Kim,Kyung Chul Moon,Da Hye Lim

doi:10.3390/diagnostics11111950

Woong Sik Jang, Hyunseul Jee + Show 7 more

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https://doi.org/10.3390/diagnostics11111950

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Journal: Diagnostics	Publication Date: Oct 20, 2021
Citations: 2	License type: CC BY 4.0

Affiliation: Korea University Medical Center

Abstract

Malaria, caused by the parasite Plasmodium and transmitted by mosquitoes, is an epidemic that mainly occurs in tropical and subtropical regions. As treatments differ across species of malarial parasites, there is a need to develop rapid diagnostic methods to differentiate malarial species. Herein, we developed a multiplex malaria Pan/Pf/Pv/actin beta loop-mediated isothermal amplification (LAMP) to diagnose Plasmodium spp., P. falciparum, and P. vivax, as well as the internal control (IC), within 40 min. The detection limits of the multiplex malaria Pan/Pf/Pv/IC LAMP were 1 × 102, 1 × 102, 1 × 102, and 1 × 103 copies/µL for four vectors, including the 18S rRNA gene (Plasmodium spp.), lactate dehydrogenase gene (P. falciparum), 16S rRNA gene (P. vivax), and human actin beta gene (IC), respectively. The performance of the LAMP assay was compared and evaluated by evaluating 208 clinical samples (118 positive and 90 negative samples) with the commercial RealStar® Malaria S&T PCR Kit 1.0. The developed multiplex malaria Pan/Pf/Pv/IC LAMP assay showed comparable sensitivity (100%) and specificity (100%) with the commercial RealStar® Malaria S&T PCR Kit 1.0 (100%). These results suggest that the multiplex malaria Pan/Pf/Pv/IC LAMP could be used as a point-of-care molecular diagnostic test for malaria.

Highlights

Malaria, which is caused by the Plasmodium protozoan and transmitted by mosquitoes, is an epidemic that predominantly occurs in tropical and subtropical regions [1]
The accurate classification of malaria species during the early stage of diagnosis is valuable for the proper treatment of infected patients and prevention of overexposure to antimalarial drugs, as treatment policies for different species tend to vary, such as artemisinin-based combination therapy (ACT) for P. falciparum and chloroquine for nonfalciparum species [7,33]
Developing multiplex loop-mediated isothermal amplification (LAMP) assays lacking non-specific signals is substantially more difficult than developing multiplex quantitative PCR (qPCR), as one LAMP assay uses six or seven primer/probe mixtures, and combinations of 2–4 LAMP sets can potentially result in the non-specific amplification of several primers

Summary

Introduction

Malaria, which is caused by the Plasmodium protozoan and transmitted by mosquitoes, is an epidemic that predominantly occurs in tropical and subtropical regions [1]. Malaria infection has mainly been reported in Africa and several other countries, and more than 40% of the world’s population remains at risk from various malarial species [2]. Plasmodium species that infect humans include Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale, and Plasmodium knowlesi [4]. Malaria infections commonly cause frequent fevers, anemia, and an enlarged spleen; treatments differ for each species [5]. P. vivax and P. ovale are treated using chloroquine antibiotics; P. falciparum, P. malariae, and P. knowlesi are treated with artemisinin antibiotics [7]. These five types of human-affecting Plasmodia are responsible for an infectious disease that can be cured if appropriate treatments are employed. It is crucial to diagnose malaria promptly during early stages and distinguish among malaria species [9]

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