Evaluation of Rapid Extraction Methods Coupled with a Recombinase Polymerase Amplification Assay for Point-of-Need Diagnosis of Post-Kala-Azar Dermal Leishmaniasis

Abstract

To detect Post-kala-azar leishmaniasis (PKDL) cases, several molecular methods with promising diagnostic efficacy have been developed that involve complicated and expensive DNA extraction methods, thus limiting their application in resource-poor settings. As an alternative, we evaluated two rapid DNA extraction methods and determined their impact on the detection of the parasite DNA using our newly developed recombinase polymerase amplification (RPA) assay. Skin samples were collected from suspected PKDL cases following their diagnosis through national guidelines. The extracted DNA from three skin biopsy samples using three different extraction methods was subjected to RPA and qPCR. The qPCR and RPA assays exhibited highest sensitivities when reference DNA extraction method using Qiagen (Q) kit was followed. In contrast, the sensitivity of the RPA assay dropped to 76.7% and 63.3%, respectively, when the boil & spin (B&S) and SpeedXtract (SE) rapid extraction methods were performed. Despite this compromised sensitivity, the B&S-RPA technique yielded an excellent agreement with both Q-qPCR (k = 0.828) and Q-RPA (k = 0.831) techniques. As expected, the reference DNA extraction method was found to be superior in terms of diagnostic efficacy. Finally, to apply the rapid DNA extraction methods in resource-constrained settings, further methodological refinement is warranted to improve DNA yield and purity through rigorous experiments.

Keywords

• post-kala-azar dermal leishmaniasis (PKDL)
• point-of-need diagnosis
• DNA extraction
• recombinase polymerase amplification (RPA)
• real-time PCR