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Field evaluation of a real time loop-mediated isothermal amplification assay (RealAmp) for malaria diagnosis in Cruzeiro do Sul, Acre, Brazil

Conventional molecular methods, such as nested polymerase chain reaction (PCR), are very sensitive for detection of malaria parasites, but require advanced laboratory equipment and trained personnel. Real-time loop-mediated isothermal amplification (RealAmp), a loop-mediated isothermal amplification...

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Autores principales: Viana, Giselle Maria Rachid, Silva-Flannery, Luciana, Lima Barbosa, Danielle Regina, Lucchi, Naomi, do Valle, Suiane Costa Negreiros, Farias, Samela, Barbalho, Nayara, Marchesini, Paola, Rossi, Juliana Chedid Nogaredi, Udhayakumar, Venkatachalam, Póvoa, Marinete Marins, de Oliveira, Alexandre Macedo
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6040774/
https://www.ncbi.nlm.nih.gov/pubmed/29995953
http://dx.doi.org/10.1371/journal.pone.0200492
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author Viana, Giselle Maria Rachid
Silva-Flannery, Luciana
Lima Barbosa, Danielle Regina
Lucchi, Naomi
do Valle, Suiane Costa Negreiros
Farias, Samela
Barbalho, Nayara
Marchesini, Paola
Rossi, Juliana Chedid Nogaredi
Udhayakumar, Venkatachalam
Póvoa, Marinete Marins
de Oliveira, Alexandre Macedo
author_facet Viana, Giselle Maria Rachid
Silva-Flannery, Luciana
Lima Barbosa, Danielle Regina
Lucchi, Naomi
do Valle, Suiane Costa Negreiros
Farias, Samela
Barbalho, Nayara
Marchesini, Paola
Rossi, Juliana Chedid Nogaredi
Udhayakumar, Venkatachalam
Póvoa, Marinete Marins
de Oliveira, Alexandre Macedo
author_sort Viana, Giselle Maria Rachid
collection PubMed
description Conventional molecular methods, such as nested polymerase chain reaction (PCR), are very sensitive for detection of malaria parasites, but require advanced laboratory equipment and trained personnel. Real-time loop-mediated isothermal amplification (RealAmp), a loop-mediated isothermal amplification-based molecular tool (LAMP), facilitates rapid target amplification at a single temperature setting, reducing the need for sophisticated equipment. We evaluated the performance of a field-adapted RealAmp assay for malaria diagnosis in Cruzeiro do Sul, Acre State, Brazil, a remote area in Brazil with limited laboratory capabilities. We enrolled 1,000 patients with fever (axillary temperature ≥ 37.5 C) or history of fever in last 24 h presenting for malaria diagnosis from February through June 2015. DNA was extracted from dried blood spots using a boil and spin method (heat treatment) at the sample processing site, and also using commercial kits at a Brazilian national reference laboratory. RealAmp was performed for Plasmodium genus, P. falciparum, and P. vivax identification. In addition, Giemsa-stained blood smears were prepared and examined by two independent well-trained study microscopists. A combination of Real-time PCR and nested PCR was used as reference test. The sensitivity and specificity of RealAmp in the field site laboratory were 94.1% (95% confidence interval [CI]: 90.1–96.8) and 83.9% (95% CI: 81.1–86.4), respectively. The sensitivity and specificity of local microscopy were 87.7% (95% CI: 82.6–91.7) and 98.9% (95% CI: 97.8–99.4), respectively, while study microscopy showed sensitivity of 96.4% (95% CI: 93.0–98.4) and specificity of 98.2% (95% CI: 97.0–99.0). None of the three tests detected 20 P. falciparum and P. vivax mixed infections identified by the reference test. Our findings highlight that it is possible to implement simple molecular tests in facilities with limited resources such as Cruzeiro do Sul in Brazil. RealAmp sensitivity was similar to that of microscopy performed by skilled professionals; both RealAmp and study microscopy performed poorly in detection of mixed infection. Attempts to develop and evaluate simpler molecular tools should continue, especially for the detection of malaria infection in remote areas.
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spelling pubmed-60407742018-07-19 Field evaluation of a real time loop-mediated isothermal amplification assay (RealAmp) for malaria diagnosis in Cruzeiro do Sul, Acre, Brazil Viana, Giselle Maria Rachid Silva-Flannery, Luciana Lima Barbosa, Danielle Regina Lucchi, Naomi do Valle, Suiane Costa Negreiros Farias, Samela Barbalho, Nayara Marchesini, Paola Rossi, Juliana Chedid Nogaredi Udhayakumar, Venkatachalam Póvoa, Marinete Marins de Oliveira, Alexandre Macedo PLoS One Research Article Conventional molecular methods, such as nested polymerase chain reaction (PCR), are very sensitive for detection of malaria parasites, but require advanced laboratory equipment and trained personnel. Real-time loop-mediated isothermal amplification (RealAmp), a loop-mediated isothermal amplification-based molecular tool (LAMP), facilitates rapid target amplification at a single temperature setting, reducing the need for sophisticated equipment. We evaluated the performance of a field-adapted RealAmp assay for malaria diagnosis in Cruzeiro do Sul, Acre State, Brazil, a remote area in Brazil with limited laboratory capabilities. We enrolled 1,000 patients with fever (axillary temperature ≥ 37.5 C) or history of fever in last 24 h presenting for malaria diagnosis from February through June 2015. DNA was extracted from dried blood spots using a boil and spin method (heat treatment) at the sample processing site, and also using commercial kits at a Brazilian national reference laboratory. RealAmp was performed for Plasmodium genus, P. falciparum, and P. vivax identification. In addition, Giemsa-stained blood smears were prepared and examined by two independent well-trained study microscopists. A combination of Real-time PCR and nested PCR was used as reference test. The sensitivity and specificity of RealAmp in the field site laboratory were 94.1% (95% confidence interval [CI]: 90.1–96.8) and 83.9% (95% CI: 81.1–86.4), respectively. The sensitivity and specificity of local microscopy were 87.7% (95% CI: 82.6–91.7) and 98.9% (95% CI: 97.8–99.4), respectively, while study microscopy showed sensitivity of 96.4% (95% CI: 93.0–98.4) and specificity of 98.2% (95% CI: 97.0–99.0). None of the three tests detected 20 P. falciparum and P. vivax mixed infections identified by the reference test. Our findings highlight that it is possible to implement simple molecular tests in facilities with limited resources such as Cruzeiro do Sul in Brazil. RealAmp sensitivity was similar to that of microscopy performed by skilled professionals; both RealAmp and study microscopy performed poorly in detection of mixed infection. Attempts to develop and evaluate simpler molecular tools should continue, especially for the detection of malaria infection in remote areas. Public Library of Science 2018-07-11 /pmc/articles/PMC6040774/ /pubmed/29995953 http://dx.doi.org/10.1371/journal.pone.0200492 Text en https://creativecommons.org/publicdomain/zero/1.0/ This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 (https://creativecommons.org/publicdomain/zero/1.0/) public domain dedication.
spellingShingle Research Article
Viana, Giselle Maria Rachid
Silva-Flannery, Luciana
Lima Barbosa, Danielle Regina
Lucchi, Naomi
do Valle, Suiane Costa Negreiros
Farias, Samela
Barbalho, Nayara
Marchesini, Paola
Rossi, Juliana Chedid Nogaredi
Udhayakumar, Venkatachalam
Póvoa, Marinete Marins
de Oliveira, Alexandre Macedo
Field evaluation of a real time loop-mediated isothermal amplification assay (RealAmp) for malaria diagnosis in Cruzeiro do Sul, Acre, Brazil
title Field evaluation of a real time loop-mediated isothermal amplification assay (RealAmp) for malaria diagnosis in Cruzeiro do Sul, Acre, Brazil
title_full Field evaluation of a real time loop-mediated isothermal amplification assay (RealAmp) for malaria diagnosis in Cruzeiro do Sul, Acre, Brazil
title_fullStr Field evaluation of a real time loop-mediated isothermal amplification assay (RealAmp) for malaria diagnosis in Cruzeiro do Sul, Acre, Brazil
title_full_unstemmed Field evaluation of a real time loop-mediated isothermal amplification assay (RealAmp) for malaria diagnosis in Cruzeiro do Sul, Acre, Brazil
title_short Field evaluation of a real time loop-mediated isothermal amplification assay (RealAmp) for malaria diagnosis in Cruzeiro do Sul, Acre, Brazil
title_sort field evaluation of a real time loop-mediated isothermal amplification assay (realamp) for malaria diagnosis in cruzeiro do sul, acre, brazil
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6040774/
https://www.ncbi.nlm.nih.gov/pubmed/29995953
http://dx.doi.org/10.1371/journal.pone.0200492
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