Combination of ultra-rapid DNA purification (PURE) and loop-mediated isothermal amplification (LAMP) for rapid detection of Trypanosoma cruzi DNA in dried blood spots.
Background Chagas disease or American trypanosomiasis, a neglected tropical disease, is a persistent Public Health problem in Latin America and other, non-endemic, countries. Point-of-care (POC) sensitive methods are still needed to improve and extend early diagnosis in acute infections such as cong...
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Online Access: | https://doi.org/10.1371/journal.pntd.0011290 https://doaj.org/article/4495de33c7d04c0eb926177a2a5b7085 |
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ftdoajarticles:oai:doaj.org/article:4495de33c7d04c0eb926177a2a5b7085 2023-06-11T04:09:57+02:00 Combination of ultra-rapid DNA purification (PURE) and loop-mediated isothermal amplification (LAMP) for rapid detection of Trypanosoma cruzi DNA in dried blood spots. Silvia A Longhi Lady J García Casares Arturo A Muñoz-Calderón Julio Alonso-Padilla Alejandro G Schijman 2023-04-01T00:00:00Z https://doi.org/10.1371/journal.pntd.0011290 https://doaj.org/article/4495de33c7d04c0eb926177a2a5b7085 EN eng Public Library of Science (PLoS) https://doi.org/10.1371/journal.pntd.0011290 https://doaj.org/toc/1935-2727 https://doaj.org/toc/1935-2735 1935-2727 1935-2735 doi:10.1371/journal.pntd.0011290 https://doaj.org/article/4495de33c7d04c0eb926177a2a5b7085 PLoS Neglected Tropical Diseases, Vol 17, Iss 4, p e0011290 (2023) Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 article 2023 ftdoajarticles https://doi.org/10.1371/journal.pntd.0011290 2023-05-07T00:31:57Z Background Chagas disease or American trypanosomiasis, a neglected tropical disease, is a persistent Public Health problem in Latin America and other, non-endemic, countries. Point-of-care (POC) sensitive methods are still needed to improve and extend early diagnosis in acute infections such as congenital Chagas disease. The objective of this study was to analytically evaluate in the lab the performance of a qualitative POC molecular test (Loop-mediated isothermal amplification (LAMP), Eiken, Japan) for rapid diagnosis of congenital Chagas disease employing FTA cards or Whatman 903 filter paper as solid supports for small-scale volumes of human blood. Methodology/principal findings We used human blood samples artificially infected with cultured T. cruzi strains to assess the analytical performance of the test in comparison with liquid blood anticoagulated with heparin. The DNA extraction process was evaluated using the ultrarapid purification system PURE manufactured by Eiken Chemical Company (Tokio, Japan) over artificially infected liquid blood or different amounts of dried blood spot (DBS) 3- and 6-mm pieces of FTA and Whatman 903 paper. LAMP was performed on a AccuBlock (LabNet, USA) heater or in the Loopamp LF-160 incubator (Eiken, Japan), and visualization of results was either done at naked eye, using the LF-160 device or P51 Molecular Fluorescence Viewer (minipcr bio, USA). Best conditions tested showed a limit of detection (LoD) with 95% accuracy (19/20 replicates) of 5 and 20 parasites/mL, respectively for heparinized fluid blood or DBS samples. FTA cards showed better specificity than Whatman 903 filter paper. Conclusions/significance Procedures to operate LAMP reactions from small volumes of fluid blood or DBS in FTA were standardized for LAMP detection of T. cruzi DNA. Our results encourage prospective studies in neonates born to seropositive women or oral Chagas disease outbreaks to operationally evaluate the method in the field. Article in Journal/Newspaper Arctic Directory of Open Access Journals: DOAJ Articles Arctic PLOS Neglected Tropical Diseases 17 4 e0011290 |
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Open Polar |
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Directory of Open Access Journals: DOAJ Articles |
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ftdoajarticles |
language |
English |
topic |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
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Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 Silvia A Longhi Lady J García Casares Arturo A Muñoz-Calderón Julio Alonso-Padilla Alejandro G Schijman Combination of ultra-rapid DNA purification (PURE) and loop-mediated isothermal amplification (LAMP) for rapid detection of Trypanosoma cruzi DNA in dried blood spots. |
topic_facet |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
description |
Background Chagas disease or American trypanosomiasis, a neglected tropical disease, is a persistent Public Health problem in Latin America and other, non-endemic, countries. Point-of-care (POC) sensitive methods are still needed to improve and extend early diagnosis in acute infections such as congenital Chagas disease. The objective of this study was to analytically evaluate in the lab the performance of a qualitative POC molecular test (Loop-mediated isothermal amplification (LAMP), Eiken, Japan) for rapid diagnosis of congenital Chagas disease employing FTA cards or Whatman 903 filter paper as solid supports for small-scale volumes of human blood. Methodology/principal findings We used human blood samples artificially infected with cultured T. cruzi strains to assess the analytical performance of the test in comparison with liquid blood anticoagulated with heparin. The DNA extraction process was evaluated using the ultrarapid purification system PURE manufactured by Eiken Chemical Company (Tokio, Japan) over artificially infected liquid blood or different amounts of dried blood spot (DBS) 3- and 6-mm pieces of FTA and Whatman 903 paper. LAMP was performed on a AccuBlock (LabNet, USA) heater or in the Loopamp LF-160 incubator (Eiken, Japan), and visualization of results was either done at naked eye, using the LF-160 device or P51 Molecular Fluorescence Viewer (minipcr bio, USA). Best conditions tested showed a limit of detection (LoD) with 95% accuracy (19/20 replicates) of 5 and 20 parasites/mL, respectively for heparinized fluid blood or DBS samples. FTA cards showed better specificity than Whatman 903 filter paper. Conclusions/significance Procedures to operate LAMP reactions from small volumes of fluid blood or DBS in FTA were standardized for LAMP detection of T. cruzi DNA. Our results encourage prospective studies in neonates born to seropositive women or oral Chagas disease outbreaks to operationally evaluate the method in the field. |
format |
Article in Journal/Newspaper |
author |
Silvia A Longhi Lady J García Casares Arturo A Muñoz-Calderón Julio Alonso-Padilla Alejandro G Schijman |
author_facet |
Silvia A Longhi Lady J García Casares Arturo A Muñoz-Calderón Julio Alonso-Padilla Alejandro G Schijman |
author_sort |
Silvia A Longhi |
title |
Combination of ultra-rapid DNA purification (PURE) and loop-mediated isothermal amplification (LAMP) for rapid detection of Trypanosoma cruzi DNA in dried blood spots. |
title_short |
Combination of ultra-rapid DNA purification (PURE) and loop-mediated isothermal amplification (LAMP) for rapid detection of Trypanosoma cruzi DNA in dried blood spots. |
title_full |
Combination of ultra-rapid DNA purification (PURE) and loop-mediated isothermal amplification (LAMP) for rapid detection of Trypanosoma cruzi DNA in dried blood spots. |
title_fullStr |
Combination of ultra-rapid DNA purification (PURE) and loop-mediated isothermal amplification (LAMP) for rapid detection of Trypanosoma cruzi DNA in dried blood spots. |
title_full_unstemmed |
Combination of ultra-rapid DNA purification (PURE) and loop-mediated isothermal amplification (LAMP) for rapid detection of Trypanosoma cruzi DNA in dried blood spots. |
title_sort |
combination of ultra-rapid dna purification (pure) and loop-mediated isothermal amplification (lamp) for rapid detection of trypanosoma cruzi dna in dried blood spots. |
publisher |
Public Library of Science (PLoS) |
publishDate |
2023 |
url |
https://doi.org/10.1371/journal.pntd.0011290 https://doaj.org/article/4495de33c7d04c0eb926177a2a5b7085 |
geographic |
Arctic |
geographic_facet |
Arctic |
genre |
Arctic |
genre_facet |
Arctic |
op_source |
PLoS Neglected Tropical Diseases, Vol 17, Iss 4, p e0011290 (2023) |
op_relation |
https://doi.org/10.1371/journal.pntd.0011290 https://doaj.org/toc/1935-2727 https://doaj.org/toc/1935-2735 1935-2727 1935-2735 doi:10.1371/journal.pntd.0011290 https://doaj.org/article/4495de33c7d04c0eb926177a2a5b7085 |
op_doi |
https://doi.org/10.1371/journal.pntd.0011290 |
container_title |
PLOS Neglected Tropical Diseases |
container_volume |
17 |
container_issue |
4 |
container_start_page |
e0011290 |
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1768383977237774336 |