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Ethics statement
All participants aged > 18 years were informed and enrolled in the study. Written informed consent was received from all participants before collecting samples and analyzing anonymously. All methods were carried out according to relevant guidelines and regulations. This study was approved by the Ethics Committee of the Royal Thai Army Medical Department (IRBRTA 952/2562).
DNA extraction and PCR amplification for clinical samples and Leishmania parasites culture
Sample preparation
Eight milliliters of EDTA anti-coagulated blood samples were collected from eligible participants visiting the HIV Clinic at Satun Hospital, Satun Province during a 6-month follow-up period to receive antiretroviral therapy (ART)15,16. The whole blood specimen was centrifuged at 900×g for 10 min to separate the plasma and buffy coat, and was then kept at −20 °C until further used in DNA extraction15,16.
Owing to rapid cell growth and differentiation of Leishmania promastigotes in nutritious Schneider’s Drosophila medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 20% heat inactivated fetal bovine serum (GE Healthcare, Chicago, IL, USA) at 26 °C, a high yield of parasites could be obtained for DNA extraction15,16. A total of 107 promastigotes of L. orientalis (MON-324; MHOM/TH/2010/ TR), L. martiniquensis (MON-229; WHOM/TH/2011/PG) and L. donovani (MHOM/ET/67/ HU3) were harvested and washed three times with phosphate buffered saline (PBS) before DNA extraction15.
DNA extraction
DNA of buffy coat samples was extracted using the Geneaid™ DNA Isolation Kit (blood) (New Taipei, Taiwan), whereas genomic DNA of each species of Leishmania parasites was extracted using the DNeasy Extraction Kit (tissue) (Qiagen, Hilden, Germany) according to manufacturer protocols15,16. The concentration and quality of the extracted DNA were measured using a Nanodrop spectrophotometer (Denovix, Wilmington, DE, USA) at 260/280 and 260/230 ratios15,16. The DNA samples were kept at −20 °C until further use (Fig. S1).
PCR amplification and DNA detection
The internal transcribed spacer (ITS1) region of the ribosomal RNA (rRNA) gene of Leishmania was amplified based on nested PCR described by Manomat et al.3 using a FlexCycler2 Thermocycler (Analytik Jena, Jena, Germany). Promastigotes’ DNA of L. martiniquensis (WHOM/TH/2011/PG) was used as a positive control. The PCR products were electrophoresis on 1.5% agarose gel and visualized using a Molecular Imager® Gel Doc™ XR + System with Imager LabTM3.0 (Bio-Rad). Purified PCR products of positive samples were sent to Bionics Co. Ltd. (Seoul, South Korea) for sequencing. The sequencing chromatograms were validated and multiple-aligned with reference Leishmania strains retrieved from GenBank using BioEdit, Version 7.0.1.
Loop-mediated isothermal amplification (LAMP) assay
LAMP amplification
The conserved 18S rRNA gene of Leishmania was amplified based on LAMP assay as described by Ruang-areerate et al.15. A total volume of 25 µL LAMP reaction was prepared. Briefly, each reaction of LAMP mixture consisted of 40 pmol of FIP and BIP primers, 10 pmol of F3 and B3 primers, 1× Thermopol buffer, 8 mM MgSO4, 1.4 mM of each dNTP (Biotechrabbit, Hennigsdorf, Germany), 0.8 M betaine (Sigma-Aldrich), 8 U of Bst DNA Polymerase Large Fragment (New England Biolabs, Ipswich, MA, USA) and 2 µL of DNA template15,16. Additionally, 1× SYBR™ Safe DNA Gel Stain (Thermo Fisher Scientific, Waltham, MA, USA) was added to each reaction mixture14 as the nonspecific fluorescent dye indicator of LAMP products. The reaction was incubated at 65 °C for 75 min and then heated at 80 °C for 10 min to inactivate the reaction.
LAMP visualization
After incubating, the LAMP amplicons of target DNA were analyzed and confirmed based on direct visual inspection of the reaction tubes using either a blue light (BL) or an ultraviolet light UV transilluminator14,15. A positive amplification showed vivid fluorescent emission, whereas no fluorescent emission was observed in the absence of amplification. To determine a mixture of various lengths of the stem-loop DNA of LAMP products, 5 µL of reaction mixture was electrophoresed on 2% agarose gel stained with SYBR™ Safe (Thermo Fisher Scientific) and visualized using an UV transilluminator15,16.
Synthesis of LAMP probe conjugated AuNPs
Biotin LAMP probe labeling
The biotin labeling LAMP probes designed by Ruang-areerate et al.15 were used to hybridize the Leishmania LAMP targets. In brief, the loop forward (LF) region of 18S rRNA LAMP amplicons located between F2 and F1C regions was chosen and designed for specific probes that were complementary to the loop region. Finally, the LF probe was modified by functionalizing with biotin at 5′ region and custom synthesized by Bionics Co. Ltd. (Seoul, South Korea).
LAMP probe conjugated AuNPs
As described by Ruang-areerate et al.15, a 40 nm particle diameter of colloidal AuNPs stabilized in phosphate buffer that had been conjugated with streptavidin; OD = 10, were commercially synthesized by Kestrel Bioscience Thailand Co. Ltd., Thailand. Previously, streptavidin-AuNPs were diluted at 1:1 with phosphate buffer and subsequently mixed with 100 µM 5′-biotin labeling LF probe at 1:10 ratio by vigorous vortex for 15 to 30 s at room temperature in the dark to obtain 1 nmole streptavidin-AuNP-5′-biotin-probes structure (OD = 0.5). The LAMP probe conjugated AuNPs were then stored in the dark at 4 °C until used15.
To detect Leishmania LAMP amplicons, the ratio of the hybridization was conducted in a total volume of 15 µL at 65 °C for 5 min15. Briefly, the LAMP probe conjugated AuNPs and LAMP reaction mixture were mixed at 1:1 ratio. The concentration of MgSO4 was used at 100 mM in a 5 µL fix volume of postamplification mixture (15 µL) to induce aggregation and to visually detect color and colloidal change. A positive result indicated that free AuNP-LAMP probe-amplicon complexes were formed in the reaction remained deep red after incubating, whereas pale purple with dark insoluble precipitate was observed in the absence of LAMP products.
Device fabrication and quantitative LAMP assay
Fabrication of distance-based paper device
The dPADs were fabricated from Whatman filter paper No. 1 (Cole-Parmer, Vernon Hills, IL, USA). Five different patterns of dPADs were designed to semi-quantify LAMP amplicons that were divided in sample and detection zones with channel length 20 mm and width 2 mm using Adobe Illustrator, Version 25.1 (Adobe Inc., San Jose, CA, USA). All designs were printed by laser printer (Xerox ColorQube 8570, Japan) on the filter paper that the hydrophobic wax ink with a width 1 mm that controlled and limited the territory flow of the LAMP reaction sample. Then dPAD was baked at 120 °C for 3 min to induce the wax to penetrate into the filter paper and then cooled at room temperature to limit the boundary of the sample and detection zones of the dPAD. The back side of the device was sealed with a stacked transparent adhesive tape to prevent any leakage of the flow samples12.
Optimization of sample volume and pattern selection
To optimize the sample volume for the dPAD, loading volume of LAMP reaction was evaluated at 0.5, 1.5, and 2.5 µL. The sample volume that provided the highest fluorescent length of reaction flow distance regarding SYBR Safe indicator was chosen. The fluorescent distance of each loading volume was undertaken in triplicate. The migratory distances of five patterns of dPADs in triplicates were compared to select the dPAD that gained the clearest fluorescent distances at all concentrations. Linear regression was performed to assess the independent association of fluorescent distance and loading volumes at different concentrations (105 and 107 parasites/mL) as well as fluorescent distance and concentrations of five patterns.
Measurement of LAMP amplicons
To semi-quantify Leishmania parasites, the LAMP reaction mixture was dropped on the sample zone of the dPAD in which the sample flowed into the detection zone and dried within 10 min at room temperature. The device was evaluated by visual inspection under the blue light (BL) so that the migratory distance of fluorescence was measured in millimeter scale.
Semi-quantitative analysis and specificity
At the mid log phase, 20 µL of Leishmania parasites were collected and resuspended with 20 µL of phosphate buffered saline (PBS) containing 0.2% glutaraldehyde (GE, Healthcare, USA). The total parasite densities (parasites/mL) were counted at 400× magnification under light microscope using a Neubauer Chamber. To calibrate semi-quantitative analysis of the dPAD, purified genomic DNA of L. orientalis were tenfold serially diluted from 106 to 103 parasites/mL using a Nanodrop spectrophotometer, and were equivalent to 1.147 µg/µL to 0.147 ng/µL, respectively. The experiments were performed in triplicate, and nuclease-free water was used as negative control.
The positive detection using the paper device was confirmed by colorimetric precipitation of specific AuNP-LAMP probes and gel electrophoresis. The optimal volume of LAMP reaction mixture was pipetted to the sample zone of the dPAD as described previously. To achieve a ready use of the readout measurement, the millimeter scale was printed on the right side of the device. Then the mean distance of all LAMP reaction mixtures including negative and tenfold serial dilution of Leishmania’ DNA samples were substituted into a linear equation of calibration curve to calculate correlation coefficient (R2) of the visual readout obtained from the semi-quantitative fluorescent distance-based paper device.
To ensure that AuNP-LAMP probes were specific to Leishmania and cross-amplification with other pathogens was unlikely, the LAMP reactions were examined against human genomic DNA extracted from buffy coat and nonLeishmania’ DNA including genomic DNA extracted from Escherichia coli, Shigella flexneri, Streptococcus pyogenes, Neisseria gonorrhea, Plasmodium falciparum, Trichomonas vaginalis, Trypanosoma evansi and Giardia intestinalis as described by Ruang-areerate et al.15. Extracted DNA of three different Leishmania species (L. orientalis, L. martiniquensis and L. donovani) were used as positive controls, and nuclease-free water was used for no template control (NTC)15.
Evaluation of sensitivity and specificity of semi-quantitative SYBR safe and AuNP-LAMP assay using dPAD
Sensitivity and specificity of SYBR safe screening, specific AuNP-LAMP probes and semi-quantitative dPAD were determined using a total of 54 genomic DNA samples from buffy coat, consisting of 22 confirmed asymptomatic VL and 32 uninfected cases. Diagnosis of VL was confirmed when nested PCR targeting the ITS1 region of rRNA gene was positive and DNA sequence of the PCR amplicons was identical to Leishmania’ DNA. Nested PCR results are considered a reference standard method due to the unavailability of any gold standard15,16,49. These data were used to validate and evaluate the sensitivity and specificity of SYBR safe, specific AuNP-LAMP probes and dPAD in one-step LAMP assay. The strength of the agreement was determined between prereaction detection, including fluorescent closed tube (SYBR safe), and postamplification detection using AuNP-LAMP probes and the dPAD. LAMP methods were assessed using the kappa statistical test at 95% confidence intervals (CI) and P-value < 0.05 was considered statistically significant. The analysis was performed using STATA, Version SE14 (Stata Corporation, College Station, TX, USA).
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