Comparison of real time and malachite-green based loop-mediated isothermal amplification assays for the detection of Plasmodium vivax and P. falciparum

Abstract

The current context of malaria elimination requires urgent development and implementation of highly sensitive and specific methods for prompt detection and treatment of malaria parasites. Such methods should overcome current delays in diagnosis, allow the detection of low-density infections and address the difficulties in accessing remote endemic communities. In this study, we assessed the performance of the RealAmp and malachite-green loop mediated isothermal amplification (MG-LAMP) methodologies, using microscopy and conventional nested-PCR as reference techniques. Both LAMP techniques were performed for Plasmodium genus, P. falciparum, and P. vivax identification using 136 whole blood samples collected from three communities located in the Peruvian Amazon basin. Turnaround time and costs of performing the LAMP assays were estimated and compared to that of microscopy and nested-PCR. Using nested-PCR as reference standard, we calculated the sensitivity, specificity and 95% confidence interval (CI) for all methods. RealAmp had a sensitivity of 92% (95% CI: 85-96.5%) and specificity of 100% (95% CI: 89.1-100%) for species detection; sensitivity and specificity of MG-LAMP were 94% (95% CI: 87.5-97.8%) and 100% (89.1-100%), respectively. Whereas microscopy showed 88.1% sensitivity (95% CI: 80.2-93.7%) and 100% specificity (95%: 89.1-100%). The turnaround time and costs of performing the LAMP assays were lower compared to those associated with nested-PCR but higher than those associated with microscopy. The two LAMP assays were shown to be more sensitive and simple to implement than microscopy. Both LAMP methodologies could be used as large-scale screening tests, but the MG-LAMP assay uses a simple, portable heat-block while the RealAmp requires a RealAmp machine or a real-time PCR machine. This makes the MG-LAMP an appropriate choice for malaria surveillance studies in endemic sites. Use of LAMP tests in active case detection of Plasmodium parasites could help to detect positive malaria cases early.

Fig 1. Map of the study sites located in the region of Loreto (green).

The insert shows the location of the three participating communities depicted with circles and the city of Iquitos in red.

Fig 2. Limit of detection (LoD) of the genus-specific LAMP assays from 10-fold and 2-fold serial dilutions of P. falciparum reference strain 3D7.

A. RealAmp amplification curves (positive) were observed from concentrations between 100,000(10⁵) and 2.5 parasites/μl dilution. B. Colorimetric MG-LAMP results can be visualized with the naked-eye, blue/green indicate positive whereas colorless negative. Pv: P. vivax control and NTC: no template control. Range 10⁵–1 = #parasites/μl.