Evaluation of rapid extraction methods coupled with Recombinase polymerase amplification assay for point-of-need diagnosis of Post-kala-azar-dermal leishmaniasis

Introduction Post kala-azar dermal leishmaniasis (PKDL) usually develops as sequelae of visceral leishmaniasis (VL) and can manifest in multiple dermatological forms. Since PKDL patients harbor Leishmania donovani parasites and can potentially trigger inter-epidemic transmission of the disease, the success of kala-azar elimination programme could be jeopardized by these cases. Although several molecular methods with promising diagnostic efficacy have been developed to detect PKDL cases, albeit complicated and expensive DNA extraction methods limit their application in resource poor settings. To address this, in comparison to a reference DNA extraction method (Qiagen), 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. Methods Thirty suspected PKDL cases were enrolled after diagnosis by clinical examination and a positive rk39 strip test. DNA was extracted from three skin biopsy samples using either a spin column-based method (Qiagen) or one of two rapid DNA extraction methods, (Boil & Spin (B&S) and SpeedXtract (SE)). RPA and qPCR were subsequently performed with the extracted samples to detect L. donovani DNA. Results Using DNA extracted by Qiagen method, the qPCR and RPA assays exhibited sensitivities of 86.7% and 93.3% respectively. In contrast, the sensitivity of RPA assay dropped to 76.7% and 63.3%, respectively, when the B&S and SE rapid extraction methods were performed. Despite this compromised sensitivity, B&S-RPA technique yielded an excellent agreement with both Q-qPCR (k = 0.828) and Q-RPA (k =0.831) techniques. Moreover, SE-RPA showed good agreement with Q-qPCR (k = 0.755), Q-RPA (k =0.692) and B&S-RPA (k =0.635) assays. As expected, with all of the three DNA extraction methods, both qPCR and RPA assay showed absolute specificity. 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. The findings of this study represent the superior diagnostic performance of Qiagen DNA extraction method over Boil&Spin (B&S) and SpeedXtract (SE) method in detecting LD DNA through RPA assay from skin biopsy of PKDL patients. We recommend, a Spin column based (Qiagen) DNA extraction method coupled with RPA assay, as a surrogate mode of diagnosis of PKDL which can be routinely performed as an alternative of qPCR. We believe our findings, and the recommendations we make from them, could help policy makers adopt a cost- effective diagnostic method for PKDL that could be implemented in resource-limited settings to help the KEP sustain their successes.


Abstract
Introduction Post kala-azar dermal leishmaniasis (PKDL) usually develops as sequelae of visceral leishmaniasis (VL) and can manifest in multiple dermatological forms. Since PKDL patients harbor Leishmania donovani parasites and can potentially trigger inter-epidemic transmission of the disease, the success of kala-azar elimination programme could be jeopardized by these cases. Although several molecular methods with promising diagnostic efficacy have been developed to detect PKDL cases, albeit complicated and expensive DNA extraction methods limit their application in resource poor settings. To address this, in comparison to a reference DNA extraction method (Qiagen), 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.
Methods Thirty suspected PKDL cases were enrolled after diagnosis by clinical examination and a positive rk39 strip test. DNA was extracted from three skin biopsy samples using either a spin column-based method (Qiagen) or one of two rapid DNA extraction methods, (Boil & Spin (B&S) and SpeedXtract (SE)). RPA and qPCR were subsequently performed with the extracted samples to detect L. donovani DNA.
Results Using DNA extracted by Qiagen method, the qPCR and RPA assays exhibited sensitivities of 86.7% and 93.3% respectively. In contrast, the sensitivity of RPA assay dropped to 76.7% and 63.3%, respectively, when the B&S and SE rapid extraction methods were performed. Despite this compromised sensitivity, B&S-RPA technique yielded an excellent agreement with both Q-qPCR (k = 0.828) and Q-RPA (k =0.831) techniques.
Moreover, SE-RPA showed good agreement with Q-qPCR (k = 0.755), Q-RPA (k =0.692) and B&S-RPA (k =0.635) assays. As expected, with all of the three DNA extraction methods, both qPCR and RPA assay showed absolute specificity.
Conclusions This study finding substantiates the superior diagnostic efficacy of Qiagen DNA extraction method over B&S and SE method in detecting LD DNA through RPA assay from skin biopsy of PKDL patients. To apply these rapid DNA extraction methods in resource-constrained settings, further methodological refinement is warranted to improve DNA yield and purity through rigorous experiments.

Background
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). PKDL usually manifests as macules (hypo-pigmented patches), papules and nodules or a combination of three known as polymorphic skin lesions mainly on the face, trunk, legs, arms and genital organ (2,3). For unknown reasons the incidence of PKDL cases with different types of lesions varies across L. donovani endemic regions (2). In Sudan, 50-60% of treated VL patients develop PKDL within 6 months, whereas, in Indian Subcontinent, PKDL is reported to develop in 5-10% VL patients within 2-4 years after treatment (3)(4)(5).
Surprisingly, the incidence rate of PKDL increases two fold within 5 years of completion of VL treatment (6). In addition, 15-20% of PKDL cases present without a documented history of VL, suggesting that these individuals may have had a prior sub-clinical L. donovani infection that was not detected (4). PKDL, unlike VL, is not life threatening if it remains untreated but PKDL patients often unfortunately experience stigma within their society (7,8). Of further concern, the Leishmania parasites harboured 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, particularly in the Indian subcontinent (9)(10)(11).
Kala-azar elimination programme (KEP) activities in the Indian subcontinent (ISC) have contributed to a remarkable decline in the incidence of kala-azar in recent years and the KEP is now considered to be in the consolidation phase. However, PKDL is identified as a potential threat to the sustained success of the programme and its ultimate goal of kala azar elimination. Proper diagnosis and management of PKDL has consequently been set as an essential component of the KEP (12,13). The control programme is facing challenges regarding early diagnosis and treatment of PKDL, however, because of its symptomatic resemblance to other skin diseases such as leprosy, vitiligo, secondary syphilis and sarcoidosis and the lack of sensitive filed-friendly diagnostic methods (14,15). The lack of awareness and poor treatment seeking behavior of PKDL patients further complicate control activities (16,17).
Currently diagnosis of PKDL relies on clinical assessments with support from parasitological approaches (2). Direct demonstration of Leishmania amastigotes in either slit skin or skin biopsy smear provides 60-100% sensitivity in nodular lesions but has poor sensitivity in macular lesions (7-50%) (18)(19)(20). Furthermore, several antibody-based serological methods such as direct agglutination test (DAT), enzyme linked immunosorbent assay (ELISA) and rK39-based rapid diagnostic tests (RDT) have been considered as ancillary diagnostic tests for PKDL diagnosis, because all of the treated VL patients give positive result for antibody based methods, even after being cured (14,21).
In contrast, molecular methods can detect L. donovani DNA, and several conventional as well as real time PCR assays have been developed with highly sensitivities and specificities for laboratory diagnosis of both VL and PKDL (18,22). These methods can help confirm the diagnosis of PKDL in 40-94% of clinically suspected individuals (4,23), and we previously developed a promising real time PCR assay for the diagnosis of PKDL that provided excellent sensitivity (91.2%) for macular PKDL cases in endemic regions of Bangladesh (18). The application of qPCR in resource limited settings, including primary and secondary health-care facilities, is however challenging because it requires a well-equipped laboratory, trained personnel and reliable storage conditions of the reagents. Therefore, the need for a user-friendly, design-locked and field-feasible diagnostic method for PKDL detection remains. On this regard, the recombinase polymerase amplification (RPA) assay has recently emerged as a novel alternative isothermal amplification technology for the detection of nucleic acid (24,25) with the potential to overcome the limitations of poor resource settings. RPA provides results faster than conventional and even real time-PCR, despite amplifying nucleic acid at a constant temperature (42 C), and requiring less expensive and simpler equipment (24). Due to the many advantages of the RPA assay, we developed a RPA assay for detection of Leishmania donovani (LD) parasites that showed absolute sensitivity and specificity in correspondence with real-time PCR (26).
In addition to the detection method, an important parameter for the optimum sensitivity of molecular diagnostic approaches is the extraction of high-quality genomic DNA extraction from clinical specimens. Generally, Spin column-based extraction method produces pure DNA but requires use of high-speed centrifugation with enhanced washing steps, which is costly and usually not feasible in field settings. On the other hand, the SpeedXtract (SE) method that has been incorporated with RPA assay in several studies involves a magnetic bead-based lysis protocol to avoid the creation of aerosols and the use of a high-speed centrifuge. The SE method has not, however, been evaluated using skin samples.
Alternatively, we found a relatively simple DNA extraction method based on an in-house lysis buffer to be suitable for Loop-mediated isothermal assays (27,28). In an effort to develop a field-friendly diagnostic algorithm for detecting L. donovani DNA in skin samples from PKDL patients we therefore assessed various nucleic acid extraction techniques in combination with an RPA assay.

Methods And Materials
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 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 preordered 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)(32)(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.

Statistical analysis:
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.

Participants' indices:
Among the 30 clinically confirmed PKDL cases recruited, 60% were male and the mean age of the participants was 26.47 ± 12.41 years. Previous history of VL was reported in 93.3% of the PKDL patients, and 23.3% were relapse cases (Table-1 Extraction method-based performance of RPA/qPCR assay: The qPCR assay detected 26 out of 30 clinically confirmed PKDL patients with a sensitivity of 86.67%, when DNA was extracted with the Spin column based (Qiagen) method.
Likewise, the RPA assay showed elevated performance with a sensitivity of 93.33% when same DNA extraction method was followed. On the other hand, when the B&S (23/30) and SE (19/30) methods were performed, the RPA assay showed compromised diagnostic efficacy with a sensitivity of 76.7 % and 63.3 % respectively (Table-2 (Table-3

) techniques.
Furthermore, SE-RPA showed significant discordance with both Q-qPCR (p = 0.02) and Q-RPA (p = 0.004) ( Table-3 (Table-2). However, the Spin column-based extraction method (Qiagen) gave most purified DNA with a mean OD 260/280 ratio of 1.85±0.09 while the SE extraction method gave least purified DNA with a mean OD 260/280 ratio of 0.77±0.22 (Table-2). To be noted, a significant difference was found between RPA positive and negative samples regarding the DNA concentration (p<0.001) and purity (p<0.0001) for B&S method which was not observed for two other methods (Figure 2A and 2B).

Comparison of assay time and cost-effectiveness:
The cost and time estimation analysis showed that the cost and time required for the Spin column based (Qiagen) DNA extraction method coupled with qPCR assay were approximately $16.5 and 17hrs respectively for each sample, whereas RPA assay has a lower cost per sample of $7.5 and requires less performance time (15hrs 20 mins). We found the B&S-RPA assay as the least expensive (app. $5) assay, whereas SE-RPA assay was less time-consuming (˜40 min) compared to Q-RPA (15 hrs 20 mins) and B&S-RPA (13 hrs) assay.

Discussion
Tremendous declines in the incidence/rate of VL and its mortality due to the initiatives of KEP indicate substantial advancement towards the elimination of kala-azar from Indian sub-continent (ISC), including Bangladesh. The regional VL initiative is actively pursuing the elimination targets by 2020 and Bangladesh has already succeeded in reaching the targets in over 90% of its endemic sub-districts (34)(35)(36). Following the success of the attack phase, the KEP has moved to the consolidation phase with the aim of identifying and controlling potential sources of L. donovani infection in endemic areas. Our recent studies have demonstrated that, in addition to VL patients PKDL cases can competently transmit the parasites to generate new VL cases (10). PKDL cases are now considered as important reservoirs for parasites and are deemed to be key contributors to interepidemic disease transmission. Therefore, early diagnosis and treatment of PKDL should be prioritized as additional means to control the transmission of Leishmania parasites and to ensure elimination is sustained. In a recent review, Zijlstra et al suggested that validation and implementation of diagnostic methods, including qPCR or isothermal amplification technique, are essential for diagnosis of PKDL to sustain the success of VL elimination efforts in the ISC (11). In addition to clinical parameters, qPCR is considered to be the most promising method for diagnosis and assessments of cure (18,22). RPA assay has, however, shown comparable diagnostic efficacy to qPCR while surpassing PCR-based molecular methods with multitude of practical and technological advantages (26). A simple and inexpensive method of DNA extraction would make RPA assay even more feasible and sustainable in poor resource settings. We therefore evaluated three DNA extraction methods together with RPA assay and compared to the reference DNA extraction method (Qiagen) coupled with the qPCR assay to determine if we could generate a field amenable and cost-effective diagnostic method for diagnosis of PKDL.
Our data indicate that, relative to other extraction methods, considerably higher detection rates are achieved for RPA (93.3%) and qPCR (86.7%) assay when DNA extraction was performed with the Spin column-based method. We found similar sensitivity (83.4%-96.1%) for qPCR in our previous study where only macular cases with low parasite burden were included (18). In the current study all nodular and mixed PKDL cases were positive in both RPA and qPCR, a result that is consistent with their higher parasite abundance (13).
These data clearly indicate the promising diagnostic efficacy of Q-RPA assay in detecting Leishmania parasites in skin biopsies from PKDL patients. However, the sensitivity of RPA assay was compromised, when the rapid DNA extraction methods such as Boil & Spin (B&S) and SpeedXtract (SE) were used. Although several studies have reported excellent performance of isothermal amplification based assays such as LAMP and RPA assay conducted with on nucleic acid extracted from whole blood sample by Boil & Spin and SpeedXtract methods (26)(27)(28), our finding is similar to that of an earlier study performed in Sri Lanka on CL patients that reported 65.5% sensitivity of SE-RPA (37).
The comparatively lower sensitivity of Boil & Spin and SpeedXtract DNA extraction methods in RPA assay might be attributed to the inherent limitations of these techniques.
Notably, the absence of washing steps and deproteination agent such as proteinase K (SpeedXtract) leads to poor quality DNA with impurities that may inhibit the activity of recombinase and polymerase enzymes in RPA assay (38). In addition, the 2.5 times lower A260/A280 ratio in SE method provides another empirical demonstration of the underperformance of RPA assay due to the protein impurities in the DNA. Moreover, we noted that skin samples were only partially digested at the end of recommended ten minutes in SE method (notably, the digestion time for two other methods was longer).
Surprisingly, we found a significant difference in A260/A280 ratio (p<0.0001) of DNA being extracted by Boil & Spin method between RPA positive and negative samples, suggesting promise for this extraction method by increasing the DNA yield. Considering the cost and time for each of the assays, we estimated that the B&S-RPA assay is 3-fold inexpensive (at $5 per sample) compared to Q-qPCR (at ~ $16.5 per sample). On the other hand, SE-RPA assay was far less time-consuming and can produce results significantly faster (within 40 min) than other combinations. Last but not the least, B&S-RPA assay showed excellent agreement with Q-RPA and Q-qPCR assays, and SE-RPA assay presented good agreement with Q-qPCR, Q-RPA and B&S-RPA assays (table-3), further indicating the promise of B&S-RPA and SE-RPA assays in detecting LD parasite from clinical samples.
The major limitation of this study is that we performed RPA and qPCR assay with DNA extracted by three different DNA Extraction methods using different skin biopsies. This might have generated some variance, and an over-or underestimation of the performance of any of the methods, as the parasites are not evenly distributed in the lesions of PKDL patients (5). The skin biopsy procedure is invasive and requires surgical set-up and this limits the ability to collect multiple biopsies without negatively impacting patient participation and use as an active field-based case detection method. Several recent studies have indicated the satisfactory diagnostic efficacies of the less invasive slit skin, micro biopsy, and fine needle biopsy methods (20,39,40). Further evidence is required, however, before these invasive sample collection procedures can be applied for diagnosis of PKDL. Another limitation to our study is that, due to ethical issues, we used buffy coat samples from cured VL patients instead of the skin biopsy samples to determine assay specificity. Our previous study provided a basis for the use of blood sample for evaluating specificity of an index method in PKDL diagnostic studies (18).
Considering the diagnostic performance, operational cost and feasibility, our data indicate that a Spin column based (Qiagen) DNA extraction method coupled with RPA assay can be routinely performed as an alternative of qPCR for diagnosis of PKDL cases. In addition, we recommend a refined B&S method as an alternate method to the reference DNA extraction method. Further modification of the SpeedXtract method to achieve better sensitivity could generate a SE-RPA assay that could be used as a point-of-contact tool for rapid diagnosis of PKDL. Further large-scale studies are both warranted and required to generate a Q-RPA assay that can be used for the molecular diagnosis of leishmaniasis involving dermatological complications.

Conclusions
The findings of this study represent the superior diagnostic performance of Qiagen DNA extraction method over Boil&Spin (B&S) and SpeedXtract (SE) method in detecting LD DNA through RPA assay from skin biopsy of PKDL patients. We recommend, a Spin column based (Qiagen) DNA extraction method coupled with RPA assay, as a surrogate mode of diagnosis of PKDL which can be routinely performed as an alternative of qPCR. We believe our findings, and the recommendations we make from them, could help policy makers adopt a cost-effective diagnostic method for PKDL that could be implemented in resource-

Consent for Publication
Not applicable

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.