Sensitive Detection of Plasmodium vivax Using a High-Throughput, Colourimetric Loop Mediated Isothermal Amplification (HtLAMP) Platform

A Potential Novel Tool for Malaria Elimination

S Britton, Q Cheng, Matthew Grigg, CB Poole, Cielo Pasay, Timothy William, K Fornace, Nicholas Anstey, Colin Sutherland, Chris Drakeley, James McCarthy

    Research output: Contribution to journalArticleResearchpeer-review

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    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.

    Original languageEnglish
    Pages (from-to)1-16
    Number of pages16
    JournalPLoS Neglected Tropical Diseases
    Volume10
    Issue number2
    DOIs
    Publication statusPublished - 2016

    Fingerprint

    Plasmodium vivax
    Malaria
    Malaysia
    Routine Diagnostic Tests
    Microscopy
    Vivax Malaria
    Polymerase Chain Reaction
    Mitochondrial Genes
    Plasmodium
    Parasitemia
    Molecular Pathology
    Multiplex Polymerase Chain Reaction
    Limit of Detection
    Parasites
    Antigens
    Infection

    Cite this

    Britton, S ; Cheng, Q ; Grigg, Matthew ; Poole, CB ; Pasay, Cielo ; William, Timothy ; Fornace, K ; Anstey, Nicholas ; Sutherland, Colin ; Drakeley, Chris ; McCarthy, James. / Sensitive Detection of Plasmodium vivax Using a High-Throughput, Colourimetric Loop Mediated Isothermal Amplification (HtLAMP) Platform : A Potential Novel Tool for Malaria Elimination. In: PLoS Neglected Tropical Diseases. 2016 ; Vol. 10, No. 2. pp. 1-16.
    @article{a0dbf4f377d345468b4fc179eaa1be93,
    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 = "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.",
    author = "S Britton and Q Cheng and Matthew Grigg and CB Poole and Cielo Pasay and Timothy William and K Fornace and Nicholas Anstey and Colin Sutherland and Chris Drakeley and James McCarthy",
    note = "Australian National Health and Medical Research Council (Program grant #1037304, Project Grant #1045156",
    year = "2016",
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    Sensitive Detection of Plasmodium vivax Using a High-Throughput, Colourimetric Loop Mediated Isothermal Amplification (HtLAMP) Platform : A Potential Novel Tool for Malaria Elimination. / Britton, S; Cheng, Q; Grigg, Matthew; Poole, CB; Pasay, Cielo; William, Timothy; Fornace, K; Anstey, Nicholas; Sutherland, Colin; Drakeley, Chris; McCarthy, James.

    In: PLoS Neglected Tropical Diseases, Vol. 10, No. 2, 2016, p. 1-16.

    Research output: Contribution to journalArticleResearchpeer-review

    TY - JOUR

    T1 - Sensitive Detection of Plasmodium vivax Using a High-Throughput, Colourimetric Loop Mediated Isothermal Amplification (HtLAMP) Platform

    T2 - A Potential Novel Tool for Malaria Elimination

    AU - Britton, S

    AU - Cheng, Q

    AU - Grigg, Matthew

    AU - Poole, CB

    AU - Pasay, Cielo

    AU - William, Timothy

    AU - Fornace, K

    AU - Anstey, Nicholas

    AU - Sutherland, Colin

    AU - Drakeley, Chris

    AU - McCarthy, James

    N1 - Australian National Health and Medical Research Council (Program grant #1037304, Project Grant #1045156

    PY - 2016

    Y1 - 2016

    N2 - 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.

    AB - 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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    U2 - 10.1371/journal.pntd.0004443

    DO - 10.1371/journal.pntd.0004443

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