Research Article: Sensitive Detection of Plasmodium vivax Using a High-Throughput, Colourimetric Loop Mediated Isothermal Amplification (HtLAMP) Platform: A Potential Novel Tool for Malaria Elimination

Date Published: February 12, 2016

Publisher: Public Library of Science

Author(s): Sumudu Britton, Qin Cheng, Matthew J. Grigg, Catherine B. Poole, Cielo Pasay, Timothy William, Kimberley Fornace, Nicholas M. Anstey, Colin J. Sutherland, Chris Drakeley, James S. McCarthy, Alyssa E. Barry.

Abstract: IntroductionPlasmodium vivax malaria has a wide geographic distribution and poses challenges to malaria elimination that are likely to be greater than those of P. falciparum. Diagnostic tools for P. vivax infection in non-reference laboratory settings are limited to microscopy and rapid diagnostic tests but these are unreliable at low parasitemia. The development and validation of a high-throughput and sensitive assay for P. vivax is a priority.MethodsA high-throughput LAMP assay targeting a P. vivax mitochondrial gene and deploying colorimetric detection in a 96-well plate format was developed and evaluated in the laboratory. Diagnostic accuracy was compared against microscopy, antigen detection tests and PCR and validated in samples from malaria patients and community controls in a district hospital setting in Sabah, Malaysia.ResultsThe high throughput LAMP-P. vivax assay (HtLAMP-Pv) performed with an estimated limit of detection of 1.4 parasites/ μL. Assay primers demonstrated cross-reactivity with P. knowlesi but not with other Plasmodium spp. Field testing of HtLAMP-Pv was conducted using 149 samples from symptomatic malaria patients (64 P. vivax, 17 P. falciparum, 56 P. knowlesi, 7 P. malariae, 1 mixed P. knowlesi/P. vivax, with 4 excluded). When compared against multiplex PCR, HtLAMP-Pv demonstrated a sensitivity for P. vivax of 95% (95% CI 87–99%); 61/64), and specificity of 100% (95% CI 86–100%); 25/25) when P. knowlesi samples were excluded. HtLAMP-Pv testing of 112 samples from asymptomatic community controls, 7 of which had submicroscopic P. vivax infections by PCR, showed a sensitivity of 71% (95% CI 29–96%; 5/7) and specificity of 93% (95% CI87-97%; 98/105).ConclusionThis novel HtLAMP-P. vivax assay has the potential to be a useful field applicable molecular diagnostic test for P. vivax infection in elimination settings.

Partial Text: Plasmodium vivax is the most geographically widespread of the Plasmodium species that infect humans [1] and can cause severe and fatal disease [2]. In the 2014 World Malaria Report it was estimated that there were 15.8 million cases of P. vivax in 2013, accounting for 47% of malaria cases outside the African region [3]. Asymptomatic sub-microscopic P. vivax infection is commonly reported in endemic countries [4–6], accounting for on average 69.5% of P. vivax infection relative to those with patent parasitaemias from community surveys [7], compared with 50.8% for P. falciparum [8]. The parasite reservoir of P. vivax is also aided by the dormant liver stage which can cause relapsing infection, with fast gametocyte production allowing transmission earlier in the course of the disease, and the development of multidrug resistance [9] posing difficulties for both clinical management and malaria elimination goals.

The clinical samples used retrospectively in this study to validate the P. vivax specific primers were obtained with ethics approval granted by the Malaysian Medical Research Ethics Committee, Menzies School of Health Research, Australia, and London School of Hygiene and Tropical Medicine, UK.

Field-applicable diagnostic tools for the detection of Plasmodium vivax are essential components for the malaria eradication agenda [44]. Given the widespread distribution and unique challenges P. vivax poses compared with P. falciparum, there is a pressing need for the development of species- specific molecular diagnostic tools. LAMP is a molecular diagnostic tool which holds much promise in terms of its ability to be deployed in non-referral laboratory settings, given its simplicity and rapid assay turnaround time, ability to be performed on crudely extracted DNA from both whole blood and filter paper and lack of expensive equipment. The colourimetric, 96 well microtitre plate-based platform for performing LAMP (HtLAMP) for the detection of Plasmodium parasites, as previously described [43], increases the throughput of the LAMP using minimal equipment. The objective of this paper was to develop and validate a P. vivax specific HtLAMP assay on this platform with good diagnostic accuracy.



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