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

Rajashree Chowdhury,Debashis Ghosh,Shomik Maruf,Rupen Nath,Md Masud-Ur-Rashid,James Baker,Dinesh Mondal,Prakash Ghosh,Md Anik Ashfaq Khan,Faria Hossain,Md Utba Bin Rashid,Proggananda Nath,Khaledul Faisal,Ahmed Abd El Wahed,Malcolm S Duthie

doi:10.3390/tropicalmed5020095

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.

Highlights

Post kala-azar dermal leishmaniasis (PKDL) is a sequelae of Leishmania donovani infection that mostly affects individuals after successful treatment for visceral leishmaniasis (VL) [1]
Zijlstra et al suggested that validation and implementation of diagnostic methods, including qPCR or isothermal amplification techniques, are essential for diagnosis of PKDL to sustain the success of VL elimination efforts in the Indian subcontinent (ISC) [11]
The findings of this study demonstrate the superior diagnostic performance of reference DNA extraction method (Qiagen) over the boil & spin (B&S) and SpeedXtract (SE) methods in detecting Leishmania donovani (LD) DNA through recombinase polymerase amplification (RPA) assay from skin biopsy of PKDL patients

Summary

Introduction

Post kala-azar dermal leishmaniasis (PKDL) is a sequelae of Leishmania donovani infection that mostly affects individuals after successful treatment for visceral leishmaniasis (VL) [1]. The Leishmania parasites harbored within skin lesions of PKDL patients serve as the known reservoir of VL, and this plays a pivotal role in their interepidemic transmission through sandfly bites, in the Indian subcontinent [9,10,11]. This vector-borne parasitic disease is anthroponotic in the Indian subcontinent, whereas animal reservoirs are responsible for disease transmission in particular endemic regions [12,13]

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Conclusion