Evaluation of a Point-of-Need Molecular Diagnostic Tool Coupled with Rapid DNA Extraction Methods for Visceral Leishmaniasis
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Published:2023-12-11
Issue:24
Volume:13
Page:3639
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ISSN:2075-4418
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Container-title:Diagnostics
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language:en
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Short-container-title:Diagnostics
Author:
Ghosh Prakash1ORCID, Chowdhury Rajashree1ORCID, Faisal Khaledul1, Khan Md. Anik Ashfaq2, Hossain Faria1, Rahat Md. Abu1ORCID, Chowdhury Md. Arko Ayon1ORCID, Mithila Nishad Tasnim1, Kamal Mostafa1, Maruf Shomik1ORCID, Nath Rupen1, Kobialka Rea Maja2, Ceruti Arianna2, Cameron Mary3ORCID, Duthie Malcolm S.4, Wahed Ahmed Abd El2ORCID, Mondal Dinesh1
Affiliation:
1. Nutrition Research Division (NRD), International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b), Dhaka 1212, Bangladesh 2. Institute of Animal Hygiene and Veterinary Public Health, Leipzig University, D-04103 Leipzig, Germany 3. London School of Hygiene and Tropical Medicine, University of London, London WC1E 7HT, UK 4. HDT Bio, Seattle, WA 98102, USA
Abstract
A rapid, cost-effective, and simple nucleic acid isolation technique coupled with a point-of-need DNA amplification assay is a desirable goal for programmatic use. For diagnosis of Visceral Leishmaniasis (VL), Recombinase Polymerase Amplification (RPA) rapid tests for the detection of Leishmania DNA are versatile and have operational advantages over qPCR. To facilitate the delivery of the RPA test at point-of-need for VL diagnosis, we compared two rapid DNA extraction methods, SwiftDx (SX) and an in-house Boil and Spin (BS) method, coupled with RPA amplification, versus more widely used methods for DNA extraction and amplification, namely Qiagen (Q) kits and qPCR, respectively. A total of 50 confirmed VL patients and 50 controls, matched for age and gender, were recruited from Mymensingh, Bangladesh, a region highly endemic for VL. Blood samples were collected from each participant and DNA was extracted using Q, SX and BS methods. Following DNA extraction, qPCR and RPA assays were performed to detect L. donovani in downstream analysis. No significant differences in sensitivity of the RPA assay were observed between DNA extraction methods, 94.00% (95% CI: 83.45–98.75%), 90% (95% CI: 78.19–96.67%), and 88% (95% CI: 75.69–95.47%) when using Q, SX, and BS, respectively. Similarly, using qPCR, no significant differences in sensitivity were obtained when using Q or SX for DNA extraction, 94.00% (95% CI: 83.45–98.75%) and 92.00% (80.77–97.78%), respectively. It is encouraging that RPA and qPCR showed excellent agreement (k: 0.919–0.980) when different extraction methods were used and that the DNA impurities using BS had no inhibitory effect on the RPA assay. Furthermore, significantly higher DNA yields were obtained using SX and BS versus Q; however, a significantly higher parasite load was detected using qPCR when DNA was extracted using Q versus SX. Considering the cost, execution time, feasibility, and performance of RPA assay, rapid extraction methods such as the Boil and Spin technique appear to have the potential for implementation in resource-limited endemic settings. Further clinical research is warranted prior to broader application.
Funder
Swedish International Development Cooperation Agency
Subject
Clinical Biochemistry
Reference34 articles.
1. Visceral leishmaniasis: What are the needs for diagnosis, treatment and control?;Chappuis;Nat. Rev. Microbiol.,2007 2. Visceral leishmaniasis: A global overview;Wamai;J. Glob. Health Sci.,2020 3. Bi, K., Chen, Y., Zhao, S., Kuang, Y., and Wu, C.-H.J. (2018). Current Visceral Leishmaniasis Research: A Research Review to Inspire Future Study. BioMed Res. Int., 2018. 4. Selvapandiyan, A., Croft, S.L., Rijal, S., Nakhasi, H.L., and Ganguly, N.K. (2019). Innovations for the elimination and control of visceral leishmaniasis. PLoS Neglected Trop. Dis., 13. 5. Diagnostics and the neglected tropical diseases roadmap: Setting the agenda for 2030;Souza;Trans. R. Soc. Trop. Med. Hyg.,2021
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