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.

A Multi-Country, Single-Blinded, Phase 2 Study to Evaluate a Point-of-Need System for Rapid Detection of Leishmaniasis and Its Implementation in Endemic Settings

“…Here of note, the recent xenodiagnoses study performed by Mondal et al showed high infectiousness of PKDL cases towards sterile sand flies which confers the presence of parasite in peripheral blood streams [ 28 ]. In addition to the variability in clinical samples, the disparate sensitivities are attributable to the different forms of PKDL cases [ 11 , 29 , 30 ].…”

“…Finally, blood samples from endemic controls were used to determine the specificity of the assays, whereas skin samples were collected from PKDL patients to determine the sensitivity. However, previous studies substantiated the specificity of the assays through using blood samples from non-VL participants [ 11 , 30 ].…”

“…However, the DNA extraction method is still challenging to perform downstream molecular analysis at point-of-care. Recently, a study by Chowdhury et al, showed the promises of a rapid extraction method for isolation of DNA from skin samples [ 30 ]. Earlier, we showed the excellent efficiency of this method in diagnosing VL from blood samples at a field site [ 21 ].…”

Point-Of-Care or Point-Of-Need Diagnostic Tests: Time to Change Outbreak Investigation and Pathogen Detection

“…Magnetic bead-based purification methods omit the need for a centrifugation step, but several pipetting steps are still essential and total hands-on time exceeds 60 min [34]. Recently, a simple one-step purification protocol was applied to the extraction nucleic acid of paratuberculosis from fecal samples [35], Leishmania from blood and skin [36,37], Ebola from saliva [38], and rabies from tissue samples [39]. This method combined detergents and heat for the lysis step and magnetic beads to capture the inhibitors.…”

One Health and Neglected Tropical Diseases—Multisectoral Solutions to Endemic Challenges

“…Finally, several of the articles in this issue provide exciting opportunities for integrating new strategic and methodological approaches to enhance One Health approaches. For instance, Chowdhury et al [ 9 ] focus on improving point-of-need diagnostics with new molecular methods for rapid detection of kala-azar leishmaniasis in resource-limited settings. Yeh et al [ 10 ] argue that genomic technologies may be able to track changes in distribution of NTDs such as schistosomiasis in the face of climate change, as well as elucidate shifting parasite-vector dynamics.…”