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. 2016 Feb 12;10(2):e0004443.
doi: 10.1371/journal.pntd.0004443. eCollection 2016 Feb.

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

Sumudu Britton  1 , Qin Cheng  2 , Matthew J Grigg  3 , Catherine B Poole  4 , Cielo Pasay  1 , Timothy William  5 , Kimberley Fornace  6 , Nicholas M Anstey  3 , Colin J Sutherland  6 , Chris Drakeley  6 , James S McCarthy  1
Affiliations

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

Sumudu Britton et al. PLoS Negl Trop Dis. .

Abstract

Introduction: Plasmodium 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.

Methods: A 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.

Results: The 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).

Conclusion: This novel HtLAMP-P. vivax assay has the potential to be a useful field applicable molecular diagnostic test for P. vivax infection in elimination settings.

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Conflict of interest statement

I have read the journal's policy and the authors of this manuscript have the following competing interests. CBP is employed by New England Biolabs and provided advice on primer development. However the company provided no financial support to the study and did not play any role in the design, conducting of study and analysis of results. This does not alter our adherence to all PLOS NTDs policies on sharing data and materials.

Figures

Fig 1
Fig 1. HtLAMP colour change associated with hydroxynaphtholblue (HNB).
Left clear, purple colour = negative and right cloudy, blue colour = positive.
Fig 2
Fig 2. P. vivax VIV2 HtLAMP primer set superimposed on alignments of P. vivax (AY598035), P. falciparum (AJ276844) and P. knowlesi (NC_007232) cox1 genes.
Fig 3
Fig 3. Estimated Plasmodium vivax cox1 copy number based on comparison with two single copy genes, pv aldolase1 and pvmdr1.
See this image and copyright information in PMC

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