title: A simple, high-throughput, colourimetric, field applicable loop-mediated isothermal amplification (HtLAMP) assay for malaria elimination.
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).
  
  
  CONCLUSIONS
  This colourimetric HtLAMP assay holds much promise as a field applicable molecular diagnostic tool for the purpose of malaria elimination.
authors: University of Queensland, Brisbane, Australia. sumudu.britton@qimrberghofer.edu.au.|QIMR Berghofer Medical Research Institute, Brisbane, Australia. sumudu.britton@qimrberghofer.edu.au.
authors: Sumudu
authors: S
authors: Britton
authors: Australian Army Malaria Institute, Brisbane, Australia. Qin.Cheng@defence.gov.au.
authors: Qin
authors: Q
authors: Cheng
authors: London School of Hygiene and Tropical Medicine, London, UK. colin.sutherland@lshtm.ac.uk.
authors: Colin J
authors: CJ
authors: Sutherland
authors: University of Queensland, Brisbane, Australia. j.mccarthy@uq.edu.au.|QIMR Berghofer Medical Research Institute, Brisbane, Australia. j.mccarthy@uq.edu.au.
authors: James S
authors: JS
authors: McCarthy
mesh_terms: D002122:Calcium Chloride; D003124:Colorimetry / Q000379:methods*; D004272:DNA, Mitochondrial / Q000097:blood / Q000235:genetics; D016054:DNA, Protozoan / Q000097:blood / Q000235:genetics; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D057230:Limit of Detection; D008278:Magnesium Sulfate; D008288:Malaria / Q000469:parasitology* / Q000517:prevention & control; D009282:Naphthalenesulfonates; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D018512:Parasitemia; D015203:Reproducibility of Results
chemical_list: D004272:DNA, Mitochondrial; D016054:DNA, Protozoan; D009282:Naphthalenesulfonates; C540250:trisodium 3-hydroxy-4-((2Z)-2-(2-oxo-4-sulfonatonaphthalen-1-ylidene)hydrazinyl)naphthalene-2,7-disulfonate; D008278:Magnesium Sulfate; D002122:Calcium Chloride