title: Sensitive Detection of Plasmodium vivax Using a High-Throughput, Colourimetric Loop Mediated Isothermal Amplification (HtLAMP) Platform: A Potential Novel Tool for Malaria Elimination.
abstract: BACKGROUND
  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).
  
  
  CONCLUSIONS
  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.
authors: University of Queensland, Brisbane, Australia and QIMR Berghofer Medical Research Institute, Brisbane, Australia.
authors: Sumudu
authors: S
authors: Britton
authors: Australian Army Malaria Institute, Brisbane, Australia.
authors: Qin
authors: Q
authors: Cheng
authors: Menzies School of Health Research and Charles Darwin University, Darwin, Australia.
authors: Matthew J
authors: MJ
authors: Grigg
authors: New England Biolabs, Ipswich, Massachusetts, United States of America.
authors: Catherine B
authors: CB
authors: Poole
authors: University of Queensland, Brisbane, Australia and QIMR Berghofer Medical Research Institute, Brisbane, Australia.
authors: Cielo
authors: C
authors: Pasay
authors: Jesselton Medical Centre, Kota Kinabalu, Sabah, Malaysia.
authors: Timothy
authors: T
authors: William
authors: London School of Hygiene and Tropical Medicine, London, United Kingdom.
authors: Kimberley
authors: K
authors: Fornace
authors: Menzies School of Health Research and Charles Darwin University, Darwin, Australia.
authors: Nicholas M
authors: NM
authors: Anstey
authors: London School of Hygiene and Tropical Medicine, London, United Kingdom.
authors: Colin J
authors: CJ
authors: Sutherland
authors: London School of Hygiene and Tropical Medicine, London, United Kingdom.
authors: Chris
authors: C
authors: Drakeley
authors: University of Queensland, Brisbane, Australia and QIMR Berghofer Medical Research Institute, Brisbane, Australia.
authors: James S
authors: JS
authors: McCarthy
mesh_terms: D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D016780:Malaria, Vivax / Q000175:diagnosis / Q000469:parasitology* / Q000517:prevention & control; D008296:Malaysia; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D010961:Plasmodium / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D010966:Plasmodium vivax / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*