A Novel Multiplex LAMP Assay for the Rapid and Accurate Diagnosis of Visceral Leishmaniasis Caused by Leishmania infantum from Iran

Visceral leishmaniosis (VL) is one of the neglected tropical diseases despite being responsible for serious clinical symptoms, some of which lead to fatal outcomes. Thus, there is a need to apply accurate, rapid, and specific diagnostic measurements in order to control the disease and reduce the mortality rate. We aimed to develop and validate a multiplex LAMP assay for the diagnosis of VL caused by Leishmania infantum (L. infantum). Moreover, a thorough assessment was conducted to determine the effectiveness of multiplex LAMP in identifying various Leishmania species, such as Leishmania tropica (L. tropica) and Leishmania major (L. major) in comparison to Leishmania infantum (L. infantum). The diagnostic performance of the multiplex LAMP method for VL was compared to each LAMP assay, real-time polymerase chain reaction (RT-qPCR), and nested PCR technique. Two separated primers were set and used in a multiplex LAMP assay which was designed based on the ITS2 (internal transcribed spacer II) and were selected on the basis of conserved and high copy number region. Multiplex LAMP primers were designed using an online tool available at https://www.primerexplorer.jp/e. The alignment was performed using MEGA5, and the primers were further adjusted utilizing GENE Runner software. All molecular methods were tested on the serial dilution of cloned plasmid containing ITS region from standard strains of L. infantum, L. tropica, and L. major. Moreover, multiplex LAMP assay was evaluated and compared based on both standard strains and 55 clinical samples from humans as well as dogs. Various approaches were applied to interpret the multiplex LAMP reaction which deciphered a higher sensitivity when compared to the RT-qPCR for L. infantum (one copy number of plasmid, equal to 0.85 femtograms (fg) of plasmid concentration, and 0.004 parasite DNA per μL) detection while these three standard strains of Leishmania were confirmed to contain 40 DNA copies using RT-qPCR. Additionally, the multiplex LAMP detection limit was approximately equivalent to RT-qPCR for L. major and L. tropica, which included 0.342 picograms (pg) and 342 femtograms (fg) of plasmid concentration, 4 × 103 and 4 × 102 copy number of plasmid, and 17.1 and 1.71 parasite DNA per μL for L. major and L. tropica, respectively. Nested PCR exhibited a lower detection limit for L. infantum of 4 × 106 plasmid copy number compared to multiplex LAMP and RT-qPCR. Multiplex LAMP has the potential for accurate and rapid detection of infectious disease, successful treatment, and finding and monitoring asymptomatic cases, especially in low-income countries.


Introduction
Leishmania infantum (L.infantum) is a eukaryotic protozoan parasite causing visceral leishmaniasis (VL), also known as kala-azar which is transmitted by the phlebotomine sandfies.VL is the most serious and lethal form of the disease afecting internal organs such as the spleen, liver, bone marrow, and lymph nodes [1][2][3].Leishmaniasis is present in 98 countries and leads to 700,000 to 1 million new cases of cutaneous leishmaniasis (CL) and 50,000 to 90,000 new cases of the more severe VL annually.If left untreated, VL can be fatal and cause approximately 20,000 to 30,000 deaths each year.A recent systematic review and metaanalysis of studies revealed that the prevalence of human VL in Iran was 0.5%.Higher rates were observed in the northern and western regions of the country, as well as among males and children under the age of 10 [4,5].Te disease is endemic in nearly 100 countries with a 50,000 to 90,000 annual incidence rate and up to 40,000 deaths per year, making the VL a seriously neglected tropical disease.VL is caused by L. infantum, L. donovani of the old world, or L. chagasi of the new world.Due to the common characteristics of symptoms associated with VL and other diseases, the disease diagnosis is intricate and time-consuming [6,7].
L. infantum causes millions of human deaths or reservoir cases, hence early and reliable diagnosis of the VL is crucial for case management, control and surveillance of the disease, initiation of therapy, and reduction of the disease incidence within endemic areas [2,6,8].Tere is a need for accurate and rapid diagnosis of VL.Currently, available approaches include parasitological, serological, and molecular methods [9,10].Tose common diagnostic techniques include in vitro cultivation and detection of the parasites in tissue aspirates which are mostly used for VL identifcation.However, these tests are time-consuming and labor working, with variable sensitivity and specifcity values.Tese carry the potential risk of fatal hemorrhage and require a high level of expertise [8,11].Serological diagnostic methods such as indirect immunofuorescence antibody test (IFAT), enzyme-linked immunosorbent assay (ELISA), recombinant antigen-based immunochromatography test (ICT), and western blotting are moderately sensitive [2,3,12].Tese techniques may give false-negative results and cross-reactions with other diseases and lack the ability to determine the current or previous infection [12].
More recently, polymerase chain reaction (PCR)-based methods have been applied for rapid and sensitive detection, identifcation, and quantifcation of various Leishmania spp.[11][12][13].Notwithstanding conventional PCR, nested PCR, or real-time PCR (RT-qPCR) targeting the Leishmania spp.genome and using diferent primer pairs for amplifcation, the RT-qPCR has appeared as a highly sensitive technique for the VL diagnosis [12,14].Nevertheless, sensitivity and specifcity of these assays depend upon diferent aspects such as the DNA extraction method, clinical material, primers which target copy numbers, and technical conditions.Moreover, the need for sophisticated expensive equipment along with technical expertise and high costs has made these assays unafordable/unavailable in deprived (low-income) regions [3,12].
Te loop-mediated isothermal amplifcation (LAMP) has been successfully applied as a rapid, sensitive, and highly efcient technique compared to the conventional PCR technique in feld-based situations [3,9,15].It is performed at isothermal amplifcation due to strand displacement activity of Bacillus stearothermophilus (Bst) DNA polymerases.High specifcity of this method is due to the use of several separate primers recognizing six (or eight) distinct regions on the target gene.Accordingly, 10 9 -10 10 copy numbers of target gene were amplifed in an hour under constant temperature (60-65 °C) [1,16].Tese primer sets include two outer (F3 and B3) primers, two inner primers (forward inner primer (FIP) and backward inner primer (BIP)), and loop primers [17].Pyrophosphate ions generated in the LAMP reaction bind to manganese ions, thus facilitating the visualization of calcein and products using green fuorescence (SYBR Green I), turbidity (spectrophotometry measurement of absorbance at 400 nm), and agarose gel electrophoresis.Because of the inhibitory and false-positive aspects of dyes, these must be added to postreaction conditions [10,18].
Multiplex LAMP was applied to the LAMP reaction because the progression of the method could potentially increase the sensitivity of the test, which means the ability to detect low levels of parasite DNA [17,19].To target genes in Leishmania spp., kinetoplastid DNA (k-DNA), small subunit rRNA (SSU rRNA), and ribosomal internal transcribed spacer (ITS) regions are usually used.ITS region is a noncoding spacer DNA placed between the 18s rRNA and 5.8s rRNA with 40 to 200 copy numbers [18,20].Teoretically, we expect that multiplex LAMP produces an overwhelming amount of DNA products, a feature that can detect even minor template DNA amounts in samples.Eventually, the sensitivity would be enhanced.Herein, we aimed to develop and validate a novel multiplex LAMP assay for the rapid and accurate diagnosis of L. infantum.Two sets of primers were selected for multiplex LAMP to target a single gene region in order to enhance the detection limit and sensitivity of clinical sample diagnosis.Each set of primers is capable of detecting six distinct regions, and the strand displacement feature allows for the replication of a large amount of DNA that can be diagnosed using turbidity and SYBR Green I. Additionally, the accuracy of using the ITS2 region as a target DNA is being evaluated for the frst time on both standard strains and clinical samples.Te ITS2 region, also known as "internal transcribed spacer 2," is a segment of the rRNA gene found in the Leishmania parasite.Due to its high variability across diferent Leishmania species, it is a valuable tool for species identifcation.Te ITS2 region has a length of approximately 50-650 base pairs and a species-specifc copy number in Leishmania [21][22][23].Also, this technique is compared with other molecular techniques, such as RT-qPCR and nested PCR, as well as simple LAMP assays to identify L. infantum that causes VL.

Materials and Methods
A schematic diagram of this study has been provided in this study as a graphical abstract.

Ethics Statement.
Te study was approved and carried out under the guidelines of the ethical review committee of Fasa University of medical science (ethical code: IR.FUMS.REC.1395.130).Consent for the inclusion of young children was obtained from parents or guardians.Additionally, ethics approval granted the permission to collect dog samples used in this study and the sampling consent of dog keepers was taken.

Clinical
Samples.Among suspected cases (n � 105), clinical samples consisting of symptomatic and asymptomatic cases were collected from southern Iran between the summer of 2019 and winter of 2020 and had previously undergone molecular testing, biopsies, and blood smear.Venous blood samples (5 mL) were taken from 15 dogs and 40 patients with the pretreatment stage of VL.Also, 50 blood samples of asymptomatic individuals from nonendemic regions were used as negative controls.All of the VL cases were L. infantum.Te verifcation of positive samples was based on the clinical symptoms, positive PCR products, and blood smear or biopsy from the spleen or bone marrow (dog's samples were biopsy from both spleen and bone marrow and human obtained from spleen biopsy).Te whole blood samples were centrifuged at 900 × g for 10 min to separate the bufy coat, and DNA was extracted from 300 μL of each bufy coat using the QIAMP Blood Mini Kit (Qiagen, Hilden, Germany) according to the manufacture's protocol.Te quality of DNA was measured using a NanoDrop spectrophotometer (BioTek, Synergy HTX, and USA) at 260/280 and 260/230 ratios and were eluted to 30 μL fnal volumes and then stored at −20 °C for further analysis.Moreover, ffteen DNA samples were extracted from canine VL or CVL dogs which were confrmed and obtained by the Shiraz parasitology department, Shiraz University of Medical Sciences.Furthermore, 50 samples were collected from healthy individuals living in a nonendemic region.
2.5.TA Cloning.Primer design for PCR cloning based on the ITS2 region was conducted using primer BLAST from NCBI online software and was confrmed by online primer3 of the NCBI (Table 1).
PCR conditions are exhibited in Table 2.
Positive PCR products were purifed and sent to U2Biol Co., Ltd., South Korea, for sequencing.Sequences were confrmed as standard strain by Bio Edit Version 7.0.1 and carried out to validate multiple alignments with reference to Leishmania strains found from the Genebank.TA cloning was accomplished by Termo Scientifc Kit (Termo Fisher, USA) and E. coli TOP10 was used as host and Ptz57R plasmid was used as a vector.Blue-white screening was done and confrmation of TA cloning was performed by colony PCR.Plasmid extraction was carried out using alkaline-lysis protocol [25].
2.6.Nested PCR.Nested primers were designed by Gene Runner and checked by mega 5 and primer3 online software of NCBI (Table 3).
Te reaction mixture included 20 μL of the total volume containing 10 μL of 2X master mix (Amplicon Red, Denmark), 1 μL of each 10 μM forward and reverse primers, ddH 2 O, and 3 μL of 50 ng/μL DNA template.Te amplifcation was conducted in the thermal cycler (BioradT100, USA) with the conditions of 95 °C for 5 min and 30 cycles of the following steps: denaturation at 95 °C for 30 secs, annealing at 54.5 °C for 20 secs, and extension at 72 °C for 30 secs.After the completion of 30 cycles, the reaction was subjected to the fnal extension at 72 °C for 10 min.Te next nested PCR step was performed under the same condition with the same reaction mixture, except that in this case, the DNA template was 1 μL of the frst nested PCR product.Te second step products were electrophoresed on 2% gel agarose.For equivalence, the molecular techniques for Leishmania spp.detection were carried out on serial dilution of the extracted plasmid.

Real-Time PCR.
Te ITS2 sequence obtained from NCBI and region-specifc primers were designed using Allele ID software and checked by primer3 of NCBI based on the ITS2 region (Table 4).
Te reaction mixture consisted of 7.5 μL of High ROX Red master mix (Amplicon, Denmark), 0.5 μL of 10 μM forward and reverse primers, and 1 μL of DNA template.Te program (absent-present) was performed on the ABI pealtime PCR, Sep one plus system, under the following conditions: initial denaturation at 95 °C for 3 min, denaturation step at 95 °C for 45 secs, annealing at 55 °C for 45 s, extension at 72 °C for 60 s, and fnal extension at 72 °C for 10 min.

LAMP and Multiplex LAMP.
Two diferent sets of LAMP primers (external primers: F3 and B3; internal primers: FIP and BIP) were designed as multiplex based on Journal of Tropical Medicine the ITS2 sequence of Leishmania species under the names LAMP1 and LAMP2.Two sets of primers were designed by Primer Explorer V5 online software (https://www.primerexplorer.jp/e).Te consensus sequences of various Leishmania species from NBI were aligned using MEGA5 software, along with our standard strains that were sequenced.We evaluated fve sets of primers proposed by Primer Explorer online tools and selected two sets based on specifc criteria.Tese criteria included their ability to detect our standard strain of L. infantum and other Leishmania infantum sequences from NCBI, nucleotide distances between primers, recognition of six distinct regions of the ITS region, and melting temperatures between 65 °C and 75 °C, replicating the whole selected region of DNA target.After the evaluation of 5 proposed sets, 2 of them met our criteria which were selected for further evaluation.With regard to avoiding self-complementarity, hairpin formation, primerdimer formation, and GC content with the 3′ of a primer ending in G or C, GENE Runner software was used and fnally these 2 sets of primers were blasted using NCBI primer blast, and the result revealed that they are specifc primers for L. infantum detection (Table 5).
Te components of the LAMP reaction mixture included 1 μL of primer mix (5 pmol of each F3, B3, and 40 pmol of each FIP and BIP), 1 μL (8 U) of Bst DNA polymerase, 2 μL of DNA sample, 12.5 μL of 2x LAMP bufer containing 1.4 mM of each dNTP, 0.8 M betaine, 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4) 2 SO 4 , 8 mM MgSO 4 , and 0.1% Tween 20.To fnd the ideal amplifcation conditions, the mixture was examined with both primer sets, and the reactions were carried out at temperatures ranging from 59 °C to 65 °C for incubation durations of 120 min in a water bath to optimize amplifcation and facilitate detection.
Te multiplex LAMP reaction was performed in 25 μL total volume reaction mixture which contained 2.5 μL of enzyme reaction bufer (New England Biolabs Inc., MA, USA), 1 μL (8U) of Bst DNA polymerase (New England Biolabs Inc., MA, USA), 1 μL of a DNA template, 8.5 μL mixture volume of 1.4 mM of dNTPs, 0.8 M Betaine, 8 mM MgSO4 and 4.5 μL of primer mix of LAMP1, and 2 sets (5 pmol of each F3, B3 and 40 pmol of each FIP and BIP).In order to establish the optimum condition of the multiplex LAMP, several assays were examined in temperatures and time ranging from 59 °C to 65 °C for 60 to 120 min, respectively.Finally, a multiplex LAMP reaction was performed at 60 °C for 2 hrs, incubated in the water bath for optimal detection of a lower concentration of the amplifed product.
2.9.LAMP and Multiplex LAMP Visual Detection.At the end of incubation time, SYBR Green I (Termo Fisher Scientifc, Grand Island, NY) diluted in 1 : 10 ratio was added to amplifed products, and an immediate color change from pink to green indicated positive result.

Nested and Real-Time PCR.
Te results presented that all the negative controls and negative samples were negative.Nested and RT-qPCR were examined on the 12-fold serial dilutions of cloned plasmids which contained the ITS2 region of L. infantum, L. major, and L. tropica strains.Te copy number threshold for strains in the nested PCR included 4 × 10 6 , plasmid copy number or 34.2 pg of plasmid concentration equal to 171 × 10 2 number of parasite per µL (tube number 4) for both L. infantum and L. tropica, and 4 × 10 7 DNA plasmid copy number equal to 0.342 ng plasmid concentration or 171 × 10 3 number of parasites per µL (tube number 3) of L. major, which were loaded in 2% gel agarose for electrophoresis to visualize the result (Figure 2).
Te study determined the copy number threshold for various strains using RT-qPCR.Te detection limit was found to be 4 × 10 1 plasmid copy number for L. infantum, L. tropica, and L. major which corresponds to 3.42 fg of plasmid concentration, equivalent to 0.171 number of parasites per µL (tube number 9).Tese results demonstrated that the RT-qPCR detection limit is 40 DNA copy numbers of cloned plasmid for all the Leishmania strains (Figure 3).

Simple LAMP.
Two sets of LAMP primers were applied on the 12-fold serial diluted plasmids of three standard strains.Te results depicted that the detection limit of the frst set of LAMP primer (LAMP1) for L. infantum was 0.342 ng plasmid concentration, 4 × 10 6 copy number of plasmid, or 171 × 10 2 number of the parasite DNA per μL (tube numbers 1-4 which have a stream of green color and tube number 5 was orange that means negative).However, the LAMP primer sets were not able to identify the other two species (L.major and L. tropica); tube number 1 was orange in color for both of them (Figure 4).Tis particular set can be used specifcally for the detection of L. infantum but with a lower detection limit of 0.342 ng.Te second set of primers (LAMP2) was used in a simple LAMP condition like LAMP1 set.Results of the LAMP2 test on L. infantum, L. major, and L. tropica included 34.2 ng, 0.342 ng, and 0.0342 ng plasmid concentration or 4 × 10 2 , 4 × 10 6 , and 4 × 10 5 plasmid copy numbers per μL or tube numbers 8, 3, and 4 (Figure 5).In comparison to RT-qPCR, the LAM2 primers have a lower detection limit.Upon evaluation using MEGA5 software and BLAST with NCBI, it was discovered that the B3 primer of the LAMP2 primers can detect a region that is conserved in L. infantum without any mismatch and can also detect L. major and L. tropica with a mismatch.Tis fnding led us to consider evaluating these two sets as multiplex LAMP.

Multiplex LAMP.
Te detection limit of the multiplex LAMP assay was assessed by performing on the 12-fold serial diluted plasmids of L. infantum, L. major, and L. tropica.Te results indicated that the detection limit of the multiplex LAMP for L. infantum was one copy number of plasmid, 0.85 fg of plasmid concentration, and 0.004 parasite DNA per μL or tube number 12 (each 200 fg of DNA plasmid is equal to one parasite based on the website calculator (https:// cels.uri.edu/gsc/cndna.html),while detection limit of RT-qPCR was 40 DNA copy numbers for all the Leishmania strains (tube number 8).It can be concluded that multiplex LAMP is more accurate than RT-qPCR for L. infantum detection.Furthermore, the detection limits of the multiplex LAMP for L. major and L. tropica were 0.342 pg, 342 fg plasmid concentration, 4 × 103, 4 × 102 copy number of plasmid and 17.1, 1.71 parasite DNA per μL, respectively (Figure 6).
To confrm the positive results of the multiplex LAMP, the negative control without DNA was loaded on 1.5% gel agarose electrophoresis.

Journal of Tropical Medicine
Clinical sample evaluation: the diagnostic sensitivity of multiplex LAMP assay and other molecular tests was investigated by examining 105 clinical samples, 50 of which were verifed as negative and 55 as positive samples.Te RT-qPCR, nested PCR, and two conventional LAMP tests, in which those primers were utilized simultaneously as multiplex LAMP, were performed to test 55 positive and 50 negative clinical samples.Notably, 35 and 50 samples were positive using the LAMP1 and LAMP2 assays, respectively.
When two sets of primers were used in a multiplex LAMP test, all 55 positive samples were accurately identifed as positive.RT-qPCR and nested PCR assays were also tested on clinical samples in which 48 and 45 out of 55 positive samples were identifed as positive in sequence for VL caused by L. infantum (Table 6).It is noticeable that 55 clinical samples were also investigated by the multiplex LAMP method and all of them were positive for the VL agent (L.infantum) (Table 6).

Discussion
Leishmaniasis is a substantially neglected tropical disease needing profound accuracy of diagnosis, particularly in the case of the VL [26].Multiplex LAMP is a recent development that enables the detection of multiple target sequences simultaneously.Tis has the potential to improve diagnostic accuracy and reduce the risk of misdiagnosis, particularly in underdeveloped and developing countries where neglected tropical diseases like leishmaniasis are prevalent [27].Te  LAMP assay system is an afordable molecular diagnostic platform for leishmaniasis, with a disruptive cost advantage of over 10 times compared to imported devices with similar capabilities [28].LAMP has been utilized for detecting various parasitic infections including malaria, Chagas disease, and schistosomiasis, in addition to leishmaniasis [29].
Te modifcation of the LAMP reaction conditions and ingredients has improved and increased the sensitivity of the diagnostic test.Te design of two primer sets for a region and simultaneous use of them in a reaction enhanced the detection range of L. infantum at 0.85 fg containing one plasmid, while in the best conditions and the best set amongst the primers herein, the detection range for L. infantum was 342 fg of DNA (4 × 10 2 copies of the plasmid).Tese results demonstrated that the multiplex LAMP had a higher detection limit for L. infantum identifcation than those for the L. tropica and L. major.In a study conducted by Karani et al., two sets of primers were designed in which the sensitivity of primer set 1 varied depending on the Leishmania species tested, with a detection limit ranging from 30 pg to 3.6 fg.Meanwhile, primer set 2 exhibited high sensitivity, although its sensitivity also varied depending on the Leishmania species tested [7].In the current investigation similar to that of Karani et al., it was found that the LAMP2 (set 2 of LAMP primers) showed greater sensitivity of 342 fg than LAMP1 (set 1 of LAMP primers) and 342 pg for detecting L. infantum, but not for the other two standard strains (L.major and L. tropica).Tis fnding led to the decision to conduct a multiplex LAMP test for improved accuracy of L. infantum detection.
Leishmaniasis is a substantial neglected tropical disease needing profound accuracy of diagnosis, particularly in the case of the VL [26].Recently, RT-qPCR was introduced as the gold standard of Leishmania spp.diagnosis, in order to compare molecular tests and further investigation toward sensitivity of molecular diagnostic approaches.Among various molecular methods for the diagnosis of leishmaniasis, RT-qPCR has exhibited high level of efciency, not only for detecting or measuring parasite load but also as a practical tool in species detection as well as epidemiological studies.A review study of Galluzzi et al. in 2018 introduced the RT-qPCR as a sensitive and specifc method for the diagnosis of leishmaniasis.However, due to the high costs of equipment posed by countries, the RT-qPCR was not considered a routine diagnostic method [30].In a study by Dixit et al., among 267 participants (197 VL and 88 controls), 98.32% and 96.59% of sensitivity and specifcity, respectively, were observed using LAMP assay in the disease diagnosis.Te LAMP assay was signifcantly more highly efcient than the rK39 antigen rapid diagnostic test (RDT) method [2].RT-qPCR and nested PCR were compared in the present study, in which both techniques were applied on standard serial diluted samples from standard strains of L. infantum, L. major, and L. tropica based on the same sequence (ITS2).It was revealed that RT-qPCR was more efcient than nested PCR (∼34.2 fg of plasmid/μL).Tis is when multiplex LAMP exhibited more efciency than RT-qPCR for L. infantum identifcation in the current study, multiplex LAMP with 0.85 fg detection limit, and RT-qPCR with 34.2 fg, while in another study, multiplex LAMP assays exhibit comparable diagnostic accuracy to conventional RT-qPCR, with high sensitivity and specifcity values [31].
One study by Abbasi et al. in 2016 deciphered a sufcient limit of detection using the LAMP method detecting approximately 0.1 pg of the Leishmania spp.DNA.Indeed, the LAMP test was about 10 4 times more sensitive than ITS-based PCR and approximately 10 3 times more sensitive than k-DNA PCR [1].Another LAMP test was performed on L. tropica, L. donovani, and L. major which reported a detection limit of 1 fg per reaction.Te reaction duration was approximately   Journal of Tropical Medicine 9 reaction.Verma et al. exhibited that the LAMP test was able to detect 64 positive blood samples out of 66 cases, with a sensitivity of 96.9% and 65 out of 67 skin samples of VL cases with a sensitivity of 97% and 8 out of 10 skin biopsy specimens with a sensitivity of 80%.Te LAMP was also introduced as a powerful and reliable tool for rapid and efective detection of VL and CL.One of the reasons for this is due to the use of four separate primers amplifying six regions of the target gene simultaneously.In addition to the primers, for the fnal identifcation of the products, they used SYBR Green I [3].In our experiment, likewise, Verma et al. designed primers for multiplex LAMP reaction which were more efcient for L. infantum identifcation than L. tropica and L. major.Te cause can be related to the diference in the sequence or copy number of the target gene in these two strains [3].It is observed that the LAMP assay was a more sensitive tool for CL detection than alternative diagnostic methods, also being more sensitive than the gold standard method when considering using minimal invasive sampling.DNA along with 18S rDNA has been used in their study when it comes to template targets [25].Also, Jang et al. in 2021 proved that the multiplex LAMP assay results were comparable to those of RT-qPCR for the diagnosis of SARS-CoV-2 [32].
In 2018, Mukhtar et al. designed a VL diagnosis kit based on the multiplex LAMP method.Te sensitivity of this kit was 97.6% and its specifcity was 99.1%.Te method contained two sets of primers which target two distinct regions (DNA k, 18s rRNA).Te reaction mixture incubated for 40 minutes at 65 °C and the fnal products were observed under an LED light.Te result showed that the multiplex LAMP is just as capable as the applied method for designing detection kits [15].On the other hand, Karani et al. designed two sets of multiplex LAMP reaction primers based on the 18s rRNA region which was used to diagnose CL, VL, and mucosal leishmaniasis.Te detection limit of this test ranged from 30 pg to 3.6 fg, depending on the leishmaniasis strains.Te use of two sets of primers improved the detection limit.Another reason for designing a multiplex LAMP based on a gene region was the detection of a wide range of Leishmania spp.using two sets of primers [7].
In another study, the LAMP assay sensitivity and specifcity included 95% and 86%, respectively, for the diagnosis of the CL and 92% and 100%, respectively, for the VL in the whole blood.It was proposed that the LAMP assay was reliable in noninvasive sampling for the leishmaniasis [26].Te portable LAMP device could detect 100 fg of L. donovani DNA in clinical samples having 100% and 98% sensitivity and specifcity, respectively [28].A systematic review and meta-analysis in 2022 revealed that the LAMP assay had signifcant higher efcacy than conventional and PCR methods in blood samples for the VL diagnosis (estimate values of > 90% and area under the curve (AUC) values > 0.96) [33].
Te present study introduced data on a developed LAMP method called multiplex LAMP as a detection kit capable of diagnosing L. infantum with a higher level of detection than those of L. major and L. tropica.Te ITS region was adopted as the target gene characterized by a high copy number and conservation among the Leishmania species [30].Two primer sets were multiplexed to ensure the enhancement of the VL detection range accompanied by the ability to detect other species (L.major and L. tropica).Te composition of the primers was appropriately performed on serial dilution to ensure a reliable and robust new multiplex technique.A highly sensitive and specifc multiplex LAMP represented a detection limit of 0.004 parasites per μL for L. infantum as well as, approximately, 17.1 and 1.71 parasites per μL for L. major and L. tropica, respectively, on the plasmid serial dilutions.In this study, the diagnostic sensitivity of multiplex LAMP assay and other molecular tests was investigated by examining 105 clinical samples, of which 50 cases were verifed as negative and 55 as positive.All clinical samples were confrmed using PCR, various existing clinical signs, and a biopsy smear.Te RT-qPCR, nested PCR, and two conventional LAMP tests, in which those primers were utilized simultaneously as multiplex LAMP, were performed to test 55 positive and 50 negative clinical samples.In any individual LAMP assay, 35 samples were positive by the LAMP1 test and 50 of them were positive by the LAMP2 test.When two sets of primers were used in a multiplex LAMP test, all 55 positive samples were accurately identifed as positive.LAMP1 and LAMP2 have a sensitivity of 63% and 100% and a specifcity of 90% and 98%, respectively.Tis is when the combined sensitivity of multiplex LAMP was 100% and its specifcity was 96%.Although the specifcity decreased at an inappreciable amount of approximately 2%, the sensitivity enhanced when the combination of two sets of primers LAMP 1 and 2 was used in a single reaction.Bioinformatics analysis using online tools for primer evaluation showed that this increase in sensitivity may be due to the reinforcement efect of the B3 primer of LAMP2.In addition to this, the multiplex LAMP assay uses several primer sets that target distinct regions of the Leishmania genome, thereby improving the sensitivity and specifcity of the assay.Tis approach to multiplexing enables the identifcation of multiple Leishmania species or strains simultaneously, facilitating diferential diagnosis and accurate identifcation of the infecting species and leprosy infection [34].Using two sets of primers simultaneously, one of them (LAMP1) specifcally recognizes L. infantum and the other one (LAMP2) detects L. infantum with a higher detection limit.Tis may signifcantly increase the sensitivity of the assay for the diagnosis of leishmaniasis, particularly of VL.Tis improved accuracy assists in preventing false-negative results, which might lead to failure of the treatment and lead to the maintenance of reservoirs [30].Tis enhanced detection limit enables the identifcation of minimal amounts of pathogens, even in viral infections like COVID-19 with samples demonstrating elevated Ct values [35].RT-qPCR and nested PCR demonstrated similar patterns to each other with 87% and 81% sensitivity and 100% specifcity sequentially.Moreover, both of them followed the homological template of clinical sample detection.In other words, RT-qPCR in spite of rapidity and accuracy in the diagnosis, it is challenging in subclinical cases, while LAMP assay can be applied for the detection of Leishmania spp.infections in both humans and reservoir animals [7].Clinical samples were collected and tested via the multiplex LAMP, which 10 Journal of Tropical Medicine proved to successfully identify all the specimens [34].Tere is a scarcity of studies focusing on the development of a multiplex LAMP based on a single region of the gene to improve not only the detection limit but also sensitivity of clinical sample diagnosis.Furthermore, this is almost the frst time that the ITS2 region was considered as a target gene and presented acceptable accuracy based on the clinical sample results.Also, this kind of multiplex LAMP accurately detected all the infected clinical samples, whereas RT-qPCR and nested PCR were not able to detect all of them considering the same conditions.Tis fnding strongly supports the potential of multiplex LAMP assay for the identifcation of L. infantum causing VL.Efective primer design is crucial for the success of LAMP assays.It is essential for the primers to be specifc in order to avoid cross-reactivity with other organisms or genes [10].Te use of multiple primers in a multiplex LAMP assay may increase the risk of nonspecifc binding and false positives [36].In addition, we encountered several challenges during our multiplex study, such as accurately setting the concentration of primers, preventing contamination, and constantly monitoring the test with negative and internal controls.We also need to have confdence in our cloning and sequencing processes to ensure accuracy.Additionally, we had to carefully collect samples from untreated patients.Although there are some limitations and difculties, LAMP has proven to be a fast, specifc, and efective method for detecting Leishmania infection.Te development of portable LAMP devices will open new avenues for diagnosing and predicting outcomes of Leishmania infections at the point of care in the future.Tis portable device has the capability to detect and amplify even very small amounts of L. donovani DNA, as low as 100 fg.Te LAMP detection system is equipped with unique features, such as a compact fuorescent detection device, which can be utilized to measure the intensity of fuorescence produced by LAMP products [28].By utilizing self-quenching and dequenching fuorogenic probes, the LAMP system could become even more versatile, leading to the development of a reliable realtime multiplex LAMP for point-of-care setting [10].

Conclusion
Our results suggested that the multiplex LAMP method with two primer sets designed for the ITS2 region was a highly specifc tool with a detection limit approximately equal to the RT-qPCR.Te multiplex LAMP design has the sensitivity to detect 0.85 fg of DNA containing 1 copy of the plasmid (200 copy numbers of ITS region which are cloned in the plasmid are considered to be 1 parasite; 1 parasite has 200 copy numbers of this gene).Results for the negative samples and controls depicted a high specifcity (96%), suggesting the multiplex LAMP as a promising platform available for the identifcation of the VL causative agent L. infantum.

2. 4 .
Negative Control.Genomic DNAs from Trypanosoma cruzi, Plasmodium vivax, and Escherichia coli were used as negative samples provided by Dr. Hatam in the parasitology department of Shiraz University of Medical Sciences.

2. 10 .
Detection Limit.Minimum or limit of detection of molecular tests was estimated using 10 folded-plasmid serial dilutions (n � 12) of L. infantum, L. tropica, and L. major which had DNA concentrations ranging from the 4 × 10 9

Figure 1 :
Figure 1: Serial dilution of extracted plasmid and the property of each tube.

Figure 5 :
Figure 5: Te result of the second set of LAMP primers (LAMP2) on L. infantum, L major, and L. tropica extracted plasmid serial dilution with each tube number feature; NC: negative control.

Figure 6 :
Figure 6: Multiplex LAMP test on standard strains of L. infantum, L. tropica, and L. major in sequence extracted plasmid serial dilution with each tube number feature.Te green district is positive and the orange district which is the last tube is negative.

Table 1 :
Primer sequences for TA cloning.

Table 3 :
Nested external and internal primers.

Table 4 :
Real-time PCR primers for Leishmania species.
2.11.Clinical Samples Analysis.After the DNA extraction from samples of VL cases given positive results by PCR, smear, and clinical symptoms, the multiplex LAMP technique was used to investigate 55 samples according to the previous conditions.Te results were further analyzed when test tubes were supplemented with CYBER green I.For comparison, PCR, real-time PCR, and LAMP methods were performed on all clinical samples.2.12.Statistical Analysis.After evaluation of clinical samples with diferent molecular methods, we used the following formula to calculate sensitivity and specifcity.Te sensitivity � number of true positives/(number of true positives + number of false negatives).Specifcity � number of true negative/(number of false positive + number of true negative).

Table 5 :
Two sets of LAMP1, 2 primers designed for multiplex LAMP.

Table 6 :
Results of testing molecular tools on the clinical samples.