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

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).

Figure 1

Study design flowchart.

Figure 2

Venn diagram represents the distribution of 46 VL cases that were detected through different DNA extraction methods in combination with RPA assay. Among 46 VL cases, 44 cases were positive using RPA assay coupled with Qiagen, Boil and Spin, and SwiftDx DNA extraction methods, whereas only 1 case was exclusively detected through Q-RPA assay.

Figure 3

Concentration of DNA in different extraction assays and differences in the parasite load in qPCR assay: (A) Concentration of DNA isolated through Spin-column (Qiagen), Boil and Spin (BS) and SwiftDx (SX) methods. (B) Distribution of parasite burden between Q-qPCR and SX-qPCR. [* represents the significance level (**** corresponds to the p value of <0.0001)].