Evaluation of the Illumigene Malaria LAMP: A Robust Molecular Diagnostic Tool for Malaria Parasites

Abstract

Isothermal nucleic acid amplification assays such as the loop mediated isothermal amplification (LAMP), are well suited for field use as they do not require thermal cyclers to amplify the DNA. To further facilitate the use of LAMP assays in remote settings, simpler sample preparation methods and lyophilized reagents are required. The performance of a commercial malaria LAMP assay (Illumigene Malaria LAMP) was evaluated using two sample preparation workflows (simple filtration prep (SFP)) and gravity-driven filtration prep (GFP)) and pre-dispensed lyophilized reagents. Laboratory and clinical samples were tested in a field laboratory in Senegal and the results independently confirmed in a reference laboratory in the U.S.A. The Illumigene Malaria LAMP assay was easily implemented in the clinical laboratory and gave similar results to a real-time PCR reference test with limits of detection of ≤2.0 parasites/μl depending on the sample preparation method used. This assay reliably detected Plasmodium sp. parasites in a simple low-tech format, providing a much needed alternative to the more complex molecular tests for malaria diagnosis.

Figure 1. Illumigene Malaria LAMP workflow.

The Illumigene Malaria LAMP assay workflows include a sample preparation step followed by an amplification step. Two sample preparation workflows were developed: one using a simple filtration sample preparation method, Fig. 1a, and the other a 7–10 minutes gravity-driven gel filtration sample preparation method, Fig. 1b. The assays are performed as described in the corresponding insert. The necessary LAMP reagents are lyophilized in the Illumigene Malaria Test Device which consist of a TEST tube with primers targeting the Plasmodium genus and a CONTROL tube with primers to detect the housekeeping human gene, used as a DNA isolation and amplification control. The runs are performed using the illumipro-10™ incubator Reader and a qualitative result (positive, negative or invalid) is printed put after the run. These figures were prepared and provided by Meridian Bioscience, Inc.

Figure 2. Graphical representation of sensitivity and specificity of microscopy, SFP and GFP compared to PET-PCR.

The sensitivity and specificity of SFP (simple filtration prep), GFP (gravity-driven prep) methods and microscopy were calculated using the PET-PCR as the reference test. The whiskers show the 95% confidence intervals for sensitivity and specificity (centre diamonds).