A simple, high-throughput, colourimetric, field applicable loop-mediated isothermal amplification (HtLAMP) assay for malaria elimination

Britton, Sumudu, Cheng, Qin, Sutherland, Colin J. and McCarthy, James S. (2015) A simple, high-throughput, colourimetric, field applicable loop-mediated isothermal amplification (HtLAMP) assay for malaria elimination. Malaria Journal, 14 335: 1-12. doi:10.1186/s12936-015-0848-3


Author Britton, Sumudu
Cheng, Qin
Sutherland, Colin J.
McCarthy, James S.
Title A simple, high-throughput, colourimetric, field applicable loop-mediated isothermal amplification (HtLAMP) assay for malaria elimination
Journal name Malaria Journal   Check publisher's open access policy
ISSN 1475-2875
Publication date 2015-08-28
Year available 2015
Sub-type Article (original research)
DOI 10.1186/s12936-015-0848-3
Open Access Status DOI
Volume 14
Issue 335
Start page 1
End page 12
Total pages 12
Place of publication London, United Kingdom
Publisher BioMed Central
Collection year 2016
Language eng
Formatted abstract
Background
To detect all malaria infections in elimination settings sensitive, high throughput and field deployable diagnostic tools are required. Loop-mediated isothermal amplification (LAMP) represents a possible field-applicable molecular diagnostic tool. However, current LAMP platforms are limited by their capacity for high throughput.

Methods
A high-throughput LAMP (HtLAMP) platform amplifying mitochondrial targets using a 96-well microtitre plate platform, processing 85 samples and 11 controls, using hydroxynaphtholblue as a colourimetric indicator was optimized for the detection of malaria parasites. Objective confirmation of visually detectable colour change results was made using a spectrophotometer. A dilution series of laboratory-cultured 3D7 Plasmodium falciparum parasites was used to determine the limit of detection of the HtLAMP assay, using P. falciparum (HtLAMP-Pf) and Plasmodium genus (HtLAMP-Pg) primers, on whole blood and filter paper, and using different DNA extraction protocols. The diagnostic accuracy of HtLAMP was validated using clinical samples from Papua New Guinea, Malaysia, Ghana and The Gambia and its field applicability was evaluated in Kota Marudu district hospital, Sabah, Malaysia.

Results
The HtLAMP assay proved to be a simple method generating a visually-detectable blue and purple colour change that could be objectively confirmed in a spectrophotometer at a wavelength of 600 nm. When compared with PCR, overall HtLAMP-Pg had a sensitivity of 98 % (n = 260/266, 95 % CI 95–99) and specificity 83 % (n = 15/18, 95 % CI 59–96). HtLAMP-Pf had a sensitivity of 97 % (n = 124/128, 95 % CI 92–99) and specificity of 96 % (n = 151/157, 95 % CI 92–99). A validation study in a regional hospital laboratory demonstrated ease of performance and interpretation of the HtLAMP assay. HtLAMP-Pf performed in this field setting had a sensitivity of 100 % (n = 17/17, 95 % CI 80–100) and specificity of 95 % (n = 123/128, 95 % CI 90–98) compared with multiplex PCR. HtLAMP-Pf also performed well on filter paper samples from asymptomatic Ghanaian children with a sensitivity of 88 % (n = 23/25, 95 % CI 69–97).

Conclusion
This colourimetric HtLAMP assay holds much promise as a field applicable molecular diagnostic tool for the purpose of malaria elimination.
Keyword Loop mediated isothermal amplification
LAMP
Elimination
High throughput
Diagnosis
Malaria
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: Official 2016 Collection
School of Medicine Publications
 
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