Point of Care Testing for Malaria Using LAMP, Loop Mediated Isothermal Amplification

For over a century the laboratory diagnosis of malaria has relied on visualization of the parasite in blood smears. But microscopy is time and labor consuming and often inaccurate due to the considerable training and experience required. About 2 decades ago, immunochromatographic rapid diagnostic tests (RDTs) were introduced, providing a simpler and more rapid point of care diagnostic. However, RDTs, like blood smears, are limited in the detection of low-density infections. Nucleic acid amplification based assays offer markedly improved sensitivity, but standard polymerase chain reaction (PCR) diagnostics require hours of processing time, sophisticated technical skill, and expensive equipment to perform. Loop mediated isothermal amplification (LAMP) may offer the best of both worlds for malaria diagnostics, with the sensitivity of PCR but speed closer to that of RDTs. Highly sensitive diagnostics may not be appropriate in all situations. Patients with symptomatic malaria generally have high-density infections that are detectable by blood smears or RDTs, and in resource-limited settings, use of a more sophisticated assay may not be practical. However, when the goal is to interrupt transmission or eliminate malaria, there is a need to detect all infections, including asymptomatic infections, which are usually of low density [1]. In these lowendemic settings, subpatent infections, or those below the detection level of microscopy or RDT, are estimated to result in 20%–50% of all transmission episodes [2]. A malaria program that is serious about elimination cannot ignore these infections [3]. LAMP was developed in 2000 as a simple method to amplify DNA with high sensitivity, specificity, efficiency, and speed under isothermal conditions [4]. LAMP uses a DNA polymerase with strand displacement properties, usually from Bacillus stearothermophilus, obviating the need for a thermocycler. The assay has high specificity because amplification only occurs when 6 separate regions of target DNA are recognized. Specifically, amplification requires 2 inner and 2 outer primers, plus 2 additional loop-primers, which anneal at the loop structure in LAMP amplicons. This assay design enhances the sensitivity of the reaction and accelerates the reaction time to less than an hour, compared to typical PCR runs of several hours. Magnesium pyrophosphate precipitates after successful amplification, causing turbidity, and enabling visual detection. Alternatively, amplification can be detected as the loss of quenching of calcein, with emission of a fluorescent signal. The polymerases used for LAMP, which are less sensitive to inhibitors present in biological samples than some PCR polymerases, allow the use of simple and rapid DNA extraction methods such as “boil and spin” [5, 6]. Initially, LAMP was applied to pathogens causing food-borne disease, and kits to detect Salmonella, Legionella, Listeria, verotoxin-producing Escherichia coli, and Campylobacter have been commercialized. Promising assays have been developed for a variety of viruses including those causing severe acute respiratory syndrome (SARS), influenza, measles, human papilloma virus disease, and mumps. Methods have also been developed for diseases of resource-limited settings, including tuberculosis, human immunodeficiency virus (HIV) infection, and African trypanosomiasis. For malaria, the first reported primer sets for LAMP targeted the 18S ribosomal RNA gene [7]. Subsequent targeting of mitochondrial DNA provided Received and accepted 23 April 2014; electronically published 1 May 2014. Correspondence: Michelle S. Hsiang, MD, Malaria Elimination Initiative, Global Health Group, Division of Pediatric Infectious Diseases, Department of Pediatrics, University of California, San Francisco, 50 Beale St, 12th fl, Box 1224, San Francisco, CA 94105 (hsiangm@peds.ucsf.edu). The Journal of Infectious Diseases 2014;210:1167–9 © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals. permissions@oup.com. DOI: 10.1093/infdis/jiu253