Advanced Multiplex Loop Mediated Isothermal Amplification (mLAMP) Combined with Lateral Flow Detection (LFD) for Rapid Detection of Two Prevalent Malaria Species in India and Melting Curve Analysis.

Nitin Bhardwaj,Sandeep Kumar,Supriya Sharma,Sarita Kumari,Anupkumar R. Anvikar,Md Zohaib Ahmed,Jaskirat Singh,Deepali Savargaonkar,Jyoti Das

doi:10.3390/diagnostics12010032

Abstract

Isothermal techniques with lateral flow detection have emerged as a point of care (POC) technique for malaria, a major parasitic disease in tropical countries such as India. Plasmodium falciparum and Plasmodium vivax are the two most prevalent malaria species found in the country. An advanced multiplex loop-mediated isothermal amplification (mLAMP) combined with a lateral flow dipstick (LFD) technique was developed for the swift and accurate detection of P. falciparum and P. vivax, overcoming the challenges of the existing RDTs (rapid diagnostic tests). A single set of LAMP primers with a biotinylated backward inner primer (BIP primer) was used for DNA amplification of both malaria species in a single tube. The amplified DNA was hybridized with fluorescein isothiocyanate (FITC) and digoxigenin-labelled DNA probes, having a complemented sequence for the P. falciparum and P. vivax genomes, respectively. A colour band appeared on two separate LFDs for P. falciparum and P. vivax upon running the hybridized solution over them. In total, 39 clinical samples were collected from ICMR-NIMR, New Delhi. Melting curve analysis, with cross primers for both species, was used to ascertain specificity, and the sensitivity was equated with a polymerase chain reaction (PCR). The results were visualized on the LFD for both species within 60 min. We found 100% sensitivity and specificity, when compared with a traditional PCR. Melting curve analysis of mLAMP revealed the lowest detection limit of 0.15 pg/μL from sample genomic DNA. The mLAMP-LFD assays could be a potential point of care (POC) tool for early diagnosis in non-laboratory conditions, with the convenience of a reduced assay time and the simple interpretation of results.

Highlights

The World Health Organization (WHO) calculated that 229 million cases of malaria occurred globally in the year 2019
The design in this study focuses on a single set of loop-mediated isothermal amplification (LAMP) primers containing four primers and two species-specific probes labelled with fluorescent dyes to counteract the overcrowding of primers in the advanced multiplex-LAMP reaction
The 39 clinical samples were subjected to malaria testing by a gold standard microscopy test in which 20 and 19 samples were found to be positive and negative, respectively, for malaria, followed by rapid diagnostic tests (RDTs), which gave 21 positives and 18 negatives

Summary

Introduction

The World Health Organization (WHO) calculated that 229 million cases of malaria occurred globally in the year 2019. Pf and Pv were roughly the most prevalent Plasmodium parasite species causing malaria in India. Malaria is diagnosed classically by microscopy, which is considered the gold standard [2], and the other leading method used is rapid diagnostic tests (RDTs). Molecular diagnosis of malaria has many paradigms, including polymerase chain reaction (PCR), quantitative PCR (qPCR) or reverse transcriptase PCR (RT-PCR), semi-nested PCR (n-PCR), multiplex PCR (m-PCR), and isothermal methods, namely, loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), etc. Microscopy requires trained workers, is tedious, and is often found to be less sensitive, when compared to other high-end methods [3]. RDTs work effectively as they are quick and give reliable results, but the Pfhrp gene deletion problem has raised a query on its use. The debate continues over the judicious use of RDTs based on the HRPII antigen [4]

Methods

Results

Conclusion