Study sites and participants:
The study entailed both field and laboratory activities. Field activities were performed at Surja Kanta Kala-azar Research Centre (SKKRC), Mymensingh, Bangladesh, a region highly endemic for VL, and Laboratory activities at Emerging Infections and Parasitology, icddr,b, Dhaka, following the approval of the International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b) Institutional Review Board (IRB) (PR–17041). In total thirty treatment seeking, suspected PKDL cases residing in the endemic zone were enrolled at Surya kanta kala-azar research centre (SKKRC), the only specialized hospital for treatment of VL, PKDL, and their associated complications. The majority of the recruited PKDL patient had a history of VL and all exhibited characteristic skin rashes. All PKDL patients were positive in rk39 RDT and were diagnosed based on clinical characteristics by the hospital physician. Following initial examination, each patient was invited to participate in the study and written informed consent was obtained from either the participant or the legal guardian of children participants before samples were collected. Following standard procedures, the study physician collected three 3 mm skin biopsy samples from each participant. Each biopsy was preserved in NET buffer for subsequent DNA extraction. All PKDL patients were referred for treatment following national guidelines and each was found to be responsive to treatment. To determine the specificity of our investigative assays thirty DNA samples were extracted from buffy coat of cured VL patients and were also subjected to laboratory analyses.
DNA extraction from clinical specimen:
DNA was isolated following three DNA extraction methods:
Spin column-based method: DNA was extracted using a QIAamp DNA tissue & blood mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol with a minor modification: skin biopsy materials were kept at 37°C overnight after addition of ATL buffer and protease K. The following day, the material was homogenized then incubated at 56°C for two hours before purification.
SpeedXtract Extraction (SE) method: A simple and rapid blood lysis protocol (SpeedXtract, Qiagen, Hilden, Germany) was modified to suit DNA extraction from skin as follows: 100 μl of Buffer SL and 30 µl of Suspension A (SpeedXtract, Qiagen, Lake Constance, Germany) was added with 3 mm skin punch biopsy in a 2 ml tube and was mixed thoroughly by vortexing for 10 seconds. Thereafter, the mix was incubated at 95°C for 10 minutes and after incubation the skin biopsy was pressed with grinding pestle and mixed by vortexing. The mix was incubated at 95°C for another 10 minutes then the tube was transferred to a magnetic stand and incubated at room temperature for 1 minute. Finally, the supernatant was carefully transferred to a new tube.
Boil & Spin (B & S) method: Skin biopsy materials were kept in 37°C for overnight after addition of an in-house prepared simple lysis buffer (400 mM NaCl, 40 mM Tris pH 6.5, 0.4% SDS) (28) following addition of protease K (Qiagen, Hilden, Germany). The following day, the skin materials were homogenized then incubated at 70°C for 15 minutes. After incubation, the mixture was vortexed, spun and incubated for 5 minutes at 95 °C before centrifugation for 3 min at 10,000g. After centrifugation, 30 μL of clear supernatant was transferred to the Dilution Tube containing 345 μL of PCR grade water.
DNA purity and concentration:
To assess the purity of each extracted DNA sample, OD values at 260nm and 280nm was measured by a Thermo Scientific Nanodrop™ 2000 Spectrophotometer (Thermo Scientific, Germany) and the ratio calculated (the standard ratio for purified DNA ranges between 1.8–2.0). Subsequently, DNA concentration/quantity was determined from the OD value at 260 nm following the standard method (29).
Molecular detection of LD-DNA:
Recombinase polymerase amplification (RPA) assay: The RPA assay was performed with the extracted DNA samples following the previously published method (26). In brief, the assay was performed in a 50μl volume using a TwistAmp exo kit (TwistAmp exo kits, TwistDx, Cambridge, UK). Master mix was prepared in a tube with 420nM of RPA primer, 120nM of RPA Probe, 1x rehydration buffer and was added to the RPA lyophilized pellet. Then, 14 mM Mg acetate was pipetted into the tube lids. Subsequently, template DNA was added to the tubes and the tube was closed and mixed well. The tubes were immediately placed into the tubescanner (Twista, TwistDx, Cambridge, UK) and incubated for 15 min at 42 °C. The emitted fluorescence signals were measured at 20s intervals. A combined threshold and first derivative analysis were used for signal interpretation. The total reaction time for RPA was approximately 20 minutes.
Real time PCR: The real time PCR was also performed by a previously published method (30). Briefly, Taqman primers and probes were designed targeting conserved region of Leishmania REPL repeats (L42486.1) specific for L. donovani and L. infantum and synthesized by Applied Biosystems(30). Briefly, a 20 μL reaction mix was prepared containing 5μL template, 10 μL of TaqMan® Gene Expression Master Mix (2X), 1 μL pre-ordered primer-probe mix and PCR grade water. Amplification was performed on a Bio-rad CFX96 icycler system with following reaction conditions: 10 min at 95° C, followed by 45 cycles of 15 seconds at 95° C and 1 min at 60° C. Samples with cycle threshold (Ct) >40 were considered negative. The total reaction time for real time PCR was approximately 120 minutes.
Reagent Cost and time analysis:
The reagent costs associated with this study were assessed similarly to previous studies (31–33). We estimated the cost of each qPCR or RPA reaction including DNA extraction for individual sample, where only the operational costs including supplies, kit and reagent cost were under consideration. Costs for infrastructure, labor, training and supervision were not included in the calculation. The time required for each assay was estimated through inclusion of sample processing time prior to each respective DNA extraction method and detection time associated with either qPCR or RPA.
Parametric and non-parametric tests were performed based on the distribution of data. Kappa and McNemar’s test were performed to determine the concordance and discordance among three extraction methods in combination with RPA assay. Standard statistical formulas were followed to determine the sensitivity and specificity of the test with 95% CI. Furthermore, Receiver operating characteristic (ROC) curve analysis was performed to determine the accuracy of each of the extraction method when coupled with RPA/qPCR assay. All statistical analyses were performed using SPSS (Version 20.0) and GraphPad Prism (Version 8.1.2). P value <0.05 was considered as statistically significant.