Evaluation of a Point-of-Need Molecular Diagnostic Tool Coupled with Rapid DNA Extraction Methods for Visceral Leishmaniasis

Prakash Ghosh; Rajashree Chowdhury; Khaledul Faisal; Md Anik Ashfaq Khan; Faria Hossain; Md Abu Rahat; Md Arko Ayon Chowdhury; Nishad Tasnim Mithila; Mostafa Kamal; Shomik Maruf; Rupen Nath; Rea Maja Kobialka; Arianna Ceruti; Mary Cameron; Malcolm S Duthie; Ahmed Abd El Wahed; Dinesh Mondal

doi:10.3390/diagnostics13243639

Evaluation of a Point-of-Need Molecular Diagnostic Tool Coupled with Rapid DNA Extraction Methods for Visceral Leishmaniasis

Diagnostics (Basel). 2023 Dec 11;13(24):3639. doi: 10.3390/diagnostics13243639.

Authors

Prakash Ghosh¹, Rajashree Chowdhury¹, Khaledul Faisal¹, Md Anik Ashfaq Khan², Faria Hossain¹, Md Abu Rahat¹, Md Arko Ayon Chowdhury¹, Nishad Tasnim Mithila¹, Mostafa Kamal¹, Shomik Maruf¹, Rupen Nath¹, Rea Maja Kobialka², Arianna Ceruti², Mary Cameron³, Malcolm S Duthie⁴, Ahmed Abd El Wahed², Dinesh Mondal¹

Affiliations

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.

Keywords: point-of-need diagnosis; rapid DNA extraction; real-time PCR; recombinase polymerase amplification (RPA); visceral leishmaniasis (VL).

Grants and funding

GR-01455/Sida