Development of Quantitative Rapid Isothermal Amplification Assay for <i>Leishmania donovani</i>

Quantification of pathogen load, although challenging, is of paramount importance for accurate diagnosis and clinical management of a range of infectious diseases in a point-of-need testing (PONT) scenario such as in resource-limited settings. We formulated a quantification approach to test the stan...

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Autores principales:	Md Anik Ashfaq Khan, Khaledul Faisal, Rajashree Chowdhury, Prakash Ghosh, Faria Hossain, Manfred Weidmann, Dinesh Mondal, Ahmed Abd El Wahed
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	isothermal amplification quantitative molecular assay recombinase polymerase amplification point-of-need testing leishmaniasis Medicine (General) R5-920
Acceso en línea:	https://doaj.org/article/fecf0bd9aa2a40388c58c5b96669a94d
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spelling	oai:doaj.org-article:fecf0bd9aa2a40388c58c5b96669a94d2021-11-25T17:20:16ZDevelopment of Quantitative Rapid Isothermal Amplification Assay for <i>Leishmania donovani</i>10.3390/diagnostics111119632075-4418https://doaj.org/article/fecf0bd9aa2a40388c58c5b96669a94d2021-10-01T00:00:00Zhttps://www.mdpi.com/2075-4418/11/11/1963https://doaj.org/toc/2075-4418Quantification of pathogen load, although challenging, is of paramount importance for accurate diagnosis and clinical management of a range of infectious diseases in a point-of-need testing (PONT) scenario such as in resource-limited settings. We formulated a quantification approach to test the standard-curve based absolute quantification ability of isothermal recombinase polymerase amplification (RPA) assay. As a test of principle, a 10-fold dilution series of <i>Leishmania donovani</i> (LD) genomic DNA prepared in nuclease-free-water (NFW), and from culture-spiked-blood (CSB) were tested, and a 15 min assay was performed. A modified algorithm was formulated to derive the detection outcome. The threshold-record times (Tr) in seconds thus obtained were plotted against the initial load of parasite genomes for log-linear regression analysis. The quantitative RPA (Q-RPA) assay was further evaluated against a LD quantitative (q)-PCR assay with DNA extracted from visceral and post-Kala-azar dermal leishmaniasis case specimens and stratified into different ranges of threshold cycle (Ct). The best-fitted regression models were found linear with mean r<sup>2</sup>/root mean square error (RMSE) values of residual points (in seconds) estimated as 0.996/8.063 and 0.992/7.46 for replicated series of NFW and CSB, respectively. In both series, the lower limit of detection reached less than 0.1 parasite genome equivalent DNA. Absolute agreement between Q-RPA and LD-qPCR was found for test positivity, and strong positive correlations were observed between the Tr and Ct values (r = 0.89; <i>p</i> < 0.0001) as well as between the absolute parasite loads (r = 0.87; <i>p</i> < 0.0001) quantified by respective assays. The findings in this very first Q-RPA assay for leishmaniasis are suggestive of its potential in monitoring LD load in clinical specimens, and the development of rapid Q-RPA assays for other infectious diseases.Md Anik Ashfaq KhanKhaledul FaisalRajashree ChowdhuryPrakash GhoshFaria HossainManfred WeidmannDinesh MondalAhmed Abd El WahedMDPI AGarticleisothermal amplificationquantitative molecular assayrecombinase polymerase amplificationpoint-of-need testingleishmaniasisMedicine (General)R5-920ENDiagnostics, Vol 11, Iss 1963, p 1963 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	isothermal amplification quantitative molecular assay recombinase polymerase amplification point-of-need testing leishmaniasis Medicine (General) R5-920
spellingShingle	isothermal amplification quantitative molecular assay recombinase polymerase amplification point-of-need testing leishmaniasis Medicine (General) R5-920 Md Anik Ashfaq Khan Khaledul Faisal Rajashree Chowdhury Prakash Ghosh Faria Hossain Manfred Weidmann Dinesh Mondal Ahmed Abd El Wahed Development of Quantitative Rapid Isothermal Amplification Assay for <i>Leishmania donovani</i>
description	Quantification of pathogen load, although challenging, is of paramount importance for accurate diagnosis and clinical management of a range of infectious diseases in a point-of-need testing (PONT) scenario such as in resource-limited settings. We formulated a quantification approach to test the standard-curve based absolute quantification ability of isothermal recombinase polymerase amplification (RPA) assay. As a test of principle, a 10-fold dilution series of <i>Leishmania donovani</i> (LD) genomic DNA prepared in nuclease-free-water (NFW), and from culture-spiked-blood (CSB) were tested, and a 15 min assay was performed. A modified algorithm was formulated to derive the detection outcome. The threshold-record times (Tr) in seconds thus obtained were plotted against the initial load of parasite genomes for log-linear regression analysis. The quantitative RPA (Q-RPA) assay was further evaluated against a LD quantitative (q)-PCR assay with DNA extracted from visceral and post-Kala-azar dermal leishmaniasis case specimens and stratified into different ranges of threshold cycle (Ct). The best-fitted regression models were found linear with mean r<sup>2</sup>/root mean square error (RMSE) values of residual points (in seconds) estimated as 0.996/8.063 and 0.992/7.46 for replicated series of NFW and CSB, respectively. In both series, the lower limit of detection reached less than 0.1 parasite genome equivalent DNA. Absolute agreement between Q-RPA and LD-qPCR was found for test positivity, and strong positive correlations were observed between the Tr and Ct values (r = 0.89; <i>p</i> < 0.0001) as well as between the absolute parasite loads (r = 0.87; <i>p</i> < 0.0001) quantified by respective assays. The findings in this very first Q-RPA assay for leishmaniasis are suggestive of its potential in monitoring LD load in clinical specimens, and the development of rapid Q-RPA assays for other infectious diseases.
format	article
author	Md Anik Ashfaq Khan Khaledul Faisal Rajashree Chowdhury Prakash Ghosh Faria Hossain Manfred Weidmann Dinesh Mondal Ahmed Abd El Wahed
author_facet	Md Anik Ashfaq Khan Khaledul Faisal Rajashree Chowdhury Prakash Ghosh Faria Hossain Manfred Weidmann Dinesh Mondal Ahmed Abd El Wahed
author_sort	Md Anik Ashfaq Khan
title	Development of Quantitative Rapid Isothermal Amplification Assay for <i>Leishmania donovani</i>
title_short	Development of Quantitative Rapid Isothermal Amplification Assay for <i>Leishmania donovani</i>
title_full	Development of Quantitative Rapid Isothermal Amplification Assay for <i>Leishmania donovani</i>
title_fullStr	Development of Quantitative Rapid Isothermal Amplification Assay for <i>Leishmania donovani</i>
title_full_unstemmed	Development of Quantitative Rapid Isothermal Amplification Assay for <i>Leishmania donovani</i>
title_sort	development of quantitative rapid isothermal amplification assay for <i>leishmania donovani</i>
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/fecf0bd9aa2a40388c58c5b96669a94d
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Development of Quantitative Rapid Isothermal Amplification Assay for <i>Leishmania donovani</i>

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