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Analytical validation of real-time quantitative PCR assays for optimum diagnosis of vivax malaria

BACKGROUND: The prompt diagnosis of plasmodial species for effective treatment prevents worsening of individual health and avoids transmission maintenance or even malaria reintroduction in areas where Plasmodium does not exist. Polymerase chain reaction (PCR) allows for the detection of parasites be...

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Autores principales: Almeida-de-Oliveira, Natália Ketrin, Moreira, Otacílio C, de Lavigne, Aline Rosa, Mendonça-Lima, Leila, Werneck, Guilherme Loureiro, Daniel-Ribeiro, Cláudio Tadeu, Ferreira-da-Cruz, Maria de Fátima
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Instituto Oswaldo Cruz, Ministério da Saúde 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6358008/
https://www.ncbi.nlm.nih.gov/pubmed/30726341
http://dx.doi.org/10.1590/0074-02760180350
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author Almeida-de-Oliveira, Natália Ketrin
Moreira, Otacílio C
de Lavigne, Aline Rosa
Mendonça-Lima, Leila
Werneck, Guilherme Loureiro
Daniel-Ribeiro, Cláudio Tadeu
Ferreira-da-Cruz, Maria de Fátima
author_facet Almeida-de-Oliveira, Natália Ketrin
Moreira, Otacílio C
de Lavigne, Aline Rosa
Mendonça-Lima, Leila
Werneck, Guilherme Loureiro
Daniel-Ribeiro, Cláudio Tadeu
Ferreira-da-Cruz, Maria de Fátima
author_sort Almeida-de-Oliveira, Natália Ketrin
collection PubMed
description BACKGROUND: The prompt diagnosis of plasmodial species for effective treatment prevents worsening of individual health and avoids transmission maintenance or even malaria reintroduction in areas where Plasmodium does not exist. Polymerase chain reaction (PCR) allows for the detection of parasites below the threshold of microscopic examination. OBJECTIVE: Our aim was to develop a real-time PCR test to reduce diagnostic errors and increase efficacy. METHODS: The lower limit of quantification and the linearity/analytical sensitivity to measure sensitivity or limit of detection (LoD) were determined. Intra-assay variations (repeatability) and alterations between assays, operators, and instruments (reproducibility) were also assessed to set precision. FINDINGS: The linearity in SYBR™ Green and TaqMan™ systems was 10(6) and 10(2) copies and analytical sensitivity 1.13 and 1.17 copies/μL, respectively. Real-time PCR was more sensitive than conventional PCR, showing a LoD of 0.01 parasite (p)/μL. Reproducibility and repeatability (precision) were 100% for up to 0.1 p/μL in SYBR™ Green and 1 p/μL in TaqMan™ and conventional PCR. CONCLUSION: Real-time PCR may replace conventional PCR in reference laboratories for P. vivax detection due to its rapidity. The TaqMan™ system is the most indicated when quantification assays are required. Performing tests in triplicate when diagnosing Plasmodium-infected-asymptomatic individuals is recommended to minimise diagnostic errors.
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spelling pubmed-63580082019-02-11 Analytical validation of real-time quantitative PCR assays for optimum diagnosis of vivax malaria Almeida-de-Oliveira, Natália Ketrin Moreira, Otacílio C de Lavigne, Aline Rosa Mendonça-Lima, Leila Werneck, Guilherme Loureiro Daniel-Ribeiro, Cláudio Tadeu Ferreira-da-Cruz, Maria de Fátima Mem Inst Oswaldo Cruz Original Article BACKGROUND: The prompt diagnosis of plasmodial species for effective treatment prevents worsening of individual health and avoids transmission maintenance or even malaria reintroduction in areas where Plasmodium does not exist. Polymerase chain reaction (PCR) allows for the detection of parasites below the threshold of microscopic examination. OBJECTIVE: Our aim was to develop a real-time PCR test to reduce diagnostic errors and increase efficacy. METHODS: The lower limit of quantification and the linearity/analytical sensitivity to measure sensitivity or limit of detection (LoD) were determined. Intra-assay variations (repeatability) and alterations between assays, operators, and instruments (reproducibility) were also assessed to set precision. FINDINGS: The linearity in SYBR™ Green and TaqMan™ systems was 10(6) and 10(2) copies and analytical sensitivity 1.13 and 1.17 copies/μL, respectively. Real-time PCR was more sensitive than conventional PCR, showing a LoD of 0.01 parasite (p)/μL. Reproducibility and repeatability (precision) were 100% for up to 0.1 p/μL in SYBR™ Green and 1 p/μL in TaqMan™ and conventional PCR. CONCLUSION: Real-time PCR may replace conventional PCR in reference laboratories for P. vivax detection due to its rapidity. The TaqMan™ system is the most indicated when quantification assays are required. Performing tests in triplicate when diagnosing Plasmodium-infected-asymptomatic individuals is recommended to minimise diagnostic errors. Instituto Oswaldo Cruz, Ministério da Saúde 2019-01-31 /pmc/articles/PMC6358008/ /pubmed/30726341 http://dx.doi.org/10.1590/0074-02760180350 Text en https://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License
spellingShingle Original Article
Almeida-de-Oliveira, Natália Ketrin
Moreira, Otacílio C
de Lavigne, Aline Rosa
Mendonça-Lima, Leila
Werneck, Guilherme Loureiro
Daniel-Ribeiro, Cláudio Tadeu
Ferreira-da-Cruz, Maria de Fátima
Analytical validation of real-time quantitative PCR assays for optimum diagnosis of vivax malaria
title Analytical validation of real-time quantitative PCR assays for optimum diagnosis of vivax malaria
title_full Analytical validation of real-time quantitative PCR assays for optimum diagnosis of vivax malaria
title_fullStr Analytical validation of real-time quantitative PCR assays for optimum diagnosis of vivax malaria
title_full_unstemmed Analytical validation of real-time quantitative PCR assays for optimum diagnosis of vivax malaria
title_short Analytical validation of real-time quantitative PCR assays for optimum diagnosis of vivax malaria
title_sort analytical validation of real-time quantitative pcr assays for optimum diagnosis of vivax malaria
topic Original Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6358008/
https://www.ncbi.nlm.nih.gov/pubmed/30726341
http://dx.doi.org/10.1590/0074-02760180350
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