An Optimized Real-Time qPCR Method for the Effective Detection of Human Malaria Infections.

Saiful Arefeen Sazed,Mohammad Golam Kibria,Mohammad Shafiul Alam

doi:10.3390/diagnostics11050736

Saiful Arefeen Sazed, Mohammad Golam Kibria + Show 1 more

Open Access

https://doi.org/10.3390/diagnostics11050736

Copy DOI

Abstract

Polymerase chain reaction, although an expensive method for the detection of human Plasmodium spp., is still considered the finest for the diagnosis of malaria. The conventional diagnostic PCR is an inexpensive process but consumes a lot of time, reagents and lacks sensitivity. On the other hand, real-time PCR assays currently being used are mostly probe-based expensive methods and sometimes not feasible to detect all the species in a single amplification reaction condition. Here we have established a real-time PCR method that is time and cost effective with a single protocol to detect and distinguish five human Plasmodium species using the existing primers efficiently. The primers used here are being used in the conventional method and the sensitivity as well as specificity of this method has also been immensely improved (100%). The lower limit of detection for Plasmodium falciparum, Plasmodium vivax and Plasmodium malariae are 0.064 parasites/µL, 1.6 parasites/µL, and 0.32 parasites/µL respectively and no cross reactivity was observed. Besides, we have analyzed melt curves that can be used for further species confirmation and validation purposes using multiplex systems. This method, therefore, can be considered as an alternative to the existing lineup for molecular diagnosis of malaria in endemic countries.

Highlights

Malaria is a deadly mosquito-borne parasitic disease with an estimated 409,000 deaths and 229 million cases in 2019 [1], most of which are children on the continent of Africa [2]
Many endemic countries are heading towards the elimination of malaria [5] and have taken initiatives to point out the burdens and solutions to those hindrances one of which is asymptomatic cases [6]
We aimed to develop a new time, cost, and process efficient approach using the existing primers for the detection and distinguishing all five human malaria-causing parasites i.e., P. falciparum, P. vivax, P. malariae (Pm), P. ovale (Po), and P. knowlesi (Pk) in a single amplification reaction condition using extracted genomic DNA

Summary

Introduction

Malaria is a deadly mosquito-borne parasitic disease with an estimated 409,000 deaths and 229 million cases in 2019 [1], most of which are children on the continent of Africa [2]. Many endemic countries are heading towards the elimination of malaria [5] and have taken initiatives to point out the burdens and solutions to those hindrances one of which is asymptomatic cases [6]. Asymptomatic cases cannot be defined by the simple parasite density it is evident that there is a clear relationship between low parasitemiae and asymptomatic burden [7]. The previous studies suggest that these asymptomatic populations are filtered out during diagnosis when the methods are RDT, microscopy, and/or even conventional PCR [8,9]. Proper diagnosis is a vital element for choosing an appropriate treatment regimen based on the nature of the infection

Objectives

Methods

Results

Discussion

Conclusion