Skip to main content
Download PDF

Establish an allele-specific real-time PCR for Leishmania species identification

Infectious Diseases of Poverty volume 11, Article number: 60 (2022) Cite this article

Abstract

Background

Leishmaniasis is a serious neglected tropical disease that may lead to life-threatening outcome, which species are closely related to clinical diagnosis and patient management. The current Leishmania species determination method is not appropriate for clinical application. New Leishmania species identification tool is needed using clinical samples directly without isolation and cultivation of parasites.

Methods

A probe-based allele-specific real-time PCR assay was established for Leishmania species identification between Leishmania donovani and L. infantum for visceral leishmaniasis (VL) and among L. major, L. tropica and L. donovani/L. infantum for cutaneous leishmaniasis (CL), targeting hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and spermidine synthase (SPDSYN) gene with their species-specific single nucleotide polymorphisms (SNPs). The limit of detection of this assay was evaluated based on 8 repeated tests with intra-assay standard deviation < 0.5 and inter-assay coefficients of variability < 5%. The specificity of this assay was tested with DNA samples obtained from Plasmodium falciparum, Toxoplasma gondii, Brucella melitensis and Orientia tsutsugamushi. Total 42 clinical specimens were used to evaluate the ability of this assay for Leishmania species identification. The phylogenetic tree was constructed using HGPRT and SPDSYN gene fragments to validate the performance of this assay.

Results

This new method was able to detect 3 and 12 parasites/reaction for VL and CL respectively, and exhibited no cross-reaction with P. falciparum, T. gondii, B. melitensis, O. tsutsugamushi and non-target species of Leishmania. Twenty-two samples from VL patients were identified as L. donovani (n = 3) and L. infantum (n = 19), and 20 specimens from CL patients were identified as L. major (n = 20), providing an agreement of 100% compared with sequencing results. For further validation, 29 sequences of HGPRT fragment from nine Leishmania species and 22 sequences from VL patients were used for phylogenetic analysis, which agreed with the results of this new method. Similar results were obtained with 43 sequences of SPDSYN fragment from 18 Leishmania species and 20 sequences from CL patients.

Conclusions

Our assay provides a rapid and accurate tool for Leishmania species identification which is applicable for species-adapted therapeutic schedule and patient management.

Graphical Abstract

Background

Leishmaniasis is a zoonotic disease caused by as many as 21 species of Leishmania, which can lead to lethal or traumatic outcome and associated social stigmatization [1]. The vectors and animal hosts of Leishmania present diversity and intersectionality, making the diseases more complicated to control. Due to the infection with different species of Leishmania, many subclinical infections have no symptoms and many patients exhibit various clinical manifestations [2, 3]. Typically, visceral leishmaniasis (VL) is caused by L. donovani and L. infantum, which is a serious infection with internal organs and bone marrow and will have fatal consequence without treatment in time [4]. Cutaneous leishmaniasis (CL) and mucosal leishmaniasis (ML) are limited to the skin and mucous membranes, and caused by different Leishmania species. CL is caused by L. major, L. tropica and L. infantum which are prevalent around the Mediterranean basin, the Middle East, the horn of Africa, and the Indian subcontinent, and L. amazonensis, L. chagasi (sometimes still referred to as L. infantum in South America), L. mexicana, L. naiffi, L. braziliensis and L. guyanensis which are prevalent around Middle and South America [5]. L. braziliensis and L. aethiopica can cause overt ML [6]. Cured VL, infected with L. infantum and L. donovani, sometimes occurs post kala-azar dermal leishmaniasis (PKDL) [7, 8].

As different Leishmania species exhibit various virulence level, genetic heterogeneity and responses to chemical drugs, the outcome tended to be better when therapy was species-directed performed [9,10,11]. For instance, L. major, L. donovani, L. braziliensis (in Guatemala) and L. tropica are more sensitive to antimony compared to L. aethiopica, L. panamensis and L. braziliensis (in Brazil). Miltefosine is an effective drug for treating CL caused by L. guyanensis, L. panamensis and L. donovani, whereas CL caused by L. infantum and L. braziliensis exhibit more resistant to it. Unlike L. tropica, L. major, L. mexicana and L. braziliensis are more susceptible for paromomycin (PM). Amphotericin B is recommended to treat CL caused by L. tropica, L. braziliensis, L. major and L. aethiopica but not for L. infantum [12,13,14,15,16]. Further, as there are co-infections with different Leishmania species, it will lead to different pathogenicity and drug sensitivity which make the treatment more complicated [17, 18]. Thus, Leishmania species identification is important in treatment and patient management, including pharmaceutical selection, appropriate treatment determination (intralesional, intramuscular, oral systemic, or parenteral) and monitoring potential infection sequelae [19,20,21,22].

Traditional Leishmania diagnostic techniques, such as microscopic examination, protozoan culture in vitro and serological immunoassay, cannot identify Leishmania species. In present clinical practice, it is still based on empirical judgment according to the information of local epidemiology. However, it could make inappropriate determination for traveler and co-infections with different species [1]. There are some techniques were developed to discriminate Leishmania species, such as sequencing of individual gene, restriction fragment length polymorphism (RFLP), high resolution melting, multilocus sequencing typing and mass spectrometry [21, 23,24,25,26,27,28,29,30]. As the World Health Organization recommended, the “gold standard” method used to identify Leishmania species is multi-site enzyme electrophoresis (MLEE), which requires culture of parasites [2]. However, some Leishmania species are difficult to culture in vitro with cumbersome experiment procedure which also makes the results among different laboratories incomparable. Although some probe based real-time PCR assays were developed for Leishmania species identification, they are mainly focused on L. mexicana, L. braziliensis, L. peruviania and L. major for CL and not suitable for other common clinical infection related species [31, 32]. Thus, for clinical applications, a tool for Leishmania species identification among common clinical pathogens, such as L. donovani, L. infantum, L. major and L. tropica, is needed to be developed using clinical samples directly without isolation and cultivation of parasites.

In this study, to identify Leishmania species, hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and spermidine synthase (SPDSYN) genes were selected from 34 housekeeping genes. Our results showed that, HGPRT gene with species-specific single nucleotide polymorphisms (SNPs) can identify parasite species between L. donovani and L. infantum for VL, and SPDSYN gene with species-specific SNPs can distinguish parasite species among L. major, L. tropica and L. donovani/L. infantum for CL. Thus, an allele-specific real-time PCR technique was established for Leishmania species identification with clinical specimens from VL and CL patients.

Material and methods

Patients and samples

A total of 42 clinical samples from patients at Beijing Friendship Hospital, Capital Medical University from July 2015 to Sep 2021 (Table 1). The bone marrow (n = 22) and skin lesion tissue (n = 20) were collected from patients with VL and CL individually for allele-specific real-time PCR testing. VL patients presented with symptoms such as fever, splenomegaly and/or hepatomegaly, Leishmania amastigotes found in their bone marrow samples under microscope or PCR positive or Leishmania parasite culture positive. CL patients appeared as ulcer and nodule/plaques features in which Leishmania amastigotes were identified under microscopy. All bone marrow and skin lesion tissue were stored at liquid nitrogen till use. DNA samples of Plasmodium falciparum, Toxoplasma gondii, Brucella melitensis and Orientia tsutsugamushi were used as non-leishmaniasis controls.

Table 1 Patients’ characteristics of Visceral Leishmaniasis and Cutaneous Leishmaniasis
Full size table

Potential target fragment selection

Out of 34 genes of Leishmania with sequence polymorphism previously published, 21 were further analyzed according to the inclusion criteria as follows: first, these gene fragments were shown as markers for the molecular characterization of Leishmania strains and species; second, they are common genetic polymorphism sites for the four species (L. donovani, L. infantum, L. major and L. tropica); third, gene fragments can obtain from NCBI database among different species and strains (Additional file 1: Table S1).

Each gene sequence among different species of Leishmania parasites were analyzed using MLSTest software (v1.0.1.23, institute de Patologia experimental Universidad Nacional de Salta Argentina, Boston, MA, USA), individually, and genes with sequence polymorphisms and species-specific SNPs were screened out (Additional file 2: Table S2). Furthermore, these sites with species-specific SNPs that can be completely distinguished Leishmania species which were selected, specifically, the optimal site that can identify species between L. donovani and L. infantum for VL and distinguishing species among L. major, L. tropica and L. donovani/L. infantum for CL were selected as targets (Figs. 1 and 2).

Fig. 1
figure 1

Alignment of HGPRT gene fragment sequences from nine Leishmania species with illustrations of primers and probes using for VL species identification between L. donovani and L. infantum. HGPRT Hypoxanthine-guanine phosphoribosyl transferase, VL Visceral leishmaniasis

Full size image
Fig. 2
figure 2

Alignment of SPDSYN sequences from 18 Leishmania species with illustrations of primers and probes using for CL species identification among L. major, L. tropica and L. donovani/infantum. SPDSYN Spermidine synthase, CL Cutaneous leishmaniasis

Full size image

Primers and probes design and plasmids construction

Twenty-nine HGPRT sequences from nine different species of Leishmania parasites and 43 SPDSYN sequences from 18 different Leishmania parasites were collected from NCBI database and aligned using BIOEDIT software (v7.0.1, Ibis Biosciences, Carlsbad, CA, USA). Primers were designed based on the conserved region of sequence and probes were designed based on regions with species-specific SNPs of HGPRT genes between L. donovani and L. infantum and species-specific SNPs of SPDSYN genes among L. major, L. tropica and L. donovani/L. infantum using PRIMER EXPRESS 3.0 (Applied Biosystems-Roche, Branchburg, America) (Table 2). The sequences of the designed primers and probes were tested against the NCBI nucleotide database using the BLASTn (Basic Local Alignment Search Tool) to confirm the species specificity.

Table 2 Sequence of primers and probes for the real-time PCR for CL and VL identification
Full size table

DNA extraction

DNA was extracted from 200 µl bone marrow or 20 mg skin lesion tissue using a DNeasy Blood & Tissue Kit (Qiagen, 69506, Hilden, Germany) according to manufacturer’s instructions and DNA was stored at – 20 ℃.

Positive control plasmid construction

The HGPRT fragment of L. donovani and L. infantum, and SPDSYN fragment of L. major and L. donovani/infantum were amplified from identified clinical specimens and the fragment purified with DNA purification kit (TIANGEN, DP214, Beijing, China). The amplified HGPRT and SPDSYN fragments were ligated into plasmid pUC19 (TAKARA, 3219, Tokyo, Japan) using EcoRI and Hind III sites, individually. The correct cloning of the desired target DNA in the recombinant plasmid was confirmed by PCR amplification and DNA sequencing. Due to lack of L. tropica parasite and clinical samples from patients with L. tropica infection, SPDSYN fragment of L. tropica was synthesized based on sequence (Accession no. KM086079) and ligated into plasmid pUC19 by Sangon Biotech Co., Ltd, and then confirmed by PCR amplification and DNA sequencing.

An allele-specific real-time PCR assay for identification of Leishmania parasites

The allele-specific real-time PCR was conducted in a 20 μl reaction volume. For VL species identification, a reaction containing 10 μl of Promega GoTaq Probe qPCR Master Mix (Promega, A6101, Madison, WI, USA), 800 nmol/l forward primer VL-HGP-F2, 800 nmol/L reverse primer VL-HGP-R1, 450 nmol/L hydrolysis Probe P-HGP-LD (5′FAM/3′MGB-NFQ) and P-HGP-LI (5′VIC/3′MGB-NFQ), individually, plus 1 μl template DNA (5–50 ng). While for CL species identification, a reaction containing 10 μl of Promega GoTaq Probe qPCR Master Mix, 300 nmol/L forward primer CL-SPD-F, 300 nmol/L reverse primer CL-SPD-R, 450 nmol/L hydrolysis probes P-SPD-LM (5′VIC/3′MGB-NFQ), P-SPD-LT (5′FAM/3′MGB-NFQ) and P-SPD-LDI (5′Texas Red/3′MGB-NFQ), respectively, plus 1 μl template DNA (5–50 ng). The reaction was performed in the Applied Biosystems 7500 Fast real-time PCR System (ABI) with 95 ℃ for 2 min followed by 40 cycles of 95 ℃ for 15 s, 62 ℃ (VL) and 58 ℃ (CL) for 50 s. Each sample was tested with replicates, the plasmid constructed above were used as positive control and reaction without template DNA (distilled water) was used as negative control in all experiments.

Analytical sensitivity and specificity of the allele specific real-time PCR for identification of Leishmania species

The limit of detection (LOD) of the allele-specific real-time PCR assay was defined as the minimum number of parasites that could be detected based on 8 repeated tests. We used cultured L. infantum promastigotes enumerated under a microscope and diluted with blood obtained from healthy volunteer as 1,000, 100, 50, 25, 12, 6, 3 or 1 parasites/μl. Total DNA was extracted from each dilution. The LOD was defined based on the experimentally derived assay precision (intra-assay SD < 0.5 and inter-assay CV < 5%). The specificity of the allele-specific real-time PCR assay was tested with other DNA samples obtained from P. falciparum, T. gondii, B. melitensis and O. tsutsugamushi.

Two plasmids HGPRT/pUC19 of L. donovani and L. infantum and three plasmids SPDSYN/pUC19 of L. major, L. tropica and L. donovani/infantum were serial dilution as 102, 103, 104, 105, 106, 107, 108, 109 copies/μl, individually. For testing the ability of identification among different species, and the PCR reaction efficiency was evaluated using single template and multiple templates, respectively.

Evaluation the performances of allele-specific real-time PCR assay for Leishmania species identification with clinical samples

Total 42 clinical specimens were tested (Table 1), including 22 bone marrow from VL patients and 20 skin lesions from CL patients. These samples were tested according to the standard procedure described above. The amplification products of 42 clinical samples were sequenced with pair ends by Sangon Biotech Co., Ltd. The results of the new method were compared with sequencing method and the consistence was evaluated.

Construction the phylogenetic tree using HGPRT and SPDSYN gene fragments to validate the performance of the new assay

Total 51 HGPRT gene sequences were used for phylogenetic tree constructed, including 29 sequences with 9 species obtained from NCBI database and 22 sequences of clinical samples from patients with VL. For construction of CL phylogenetic tree, total 63 SPDSYN gene sequences were used, containing 43 SPDSYN gene sequences with 18 species obtained from NCBI database and 20 sequences of clinical samples from patients with CL. Using MEGA 7.0 software (Mega Limited, Auckland, New Zealand) to build N-J (Neighbor Joining) evolutionary tree based on Kimura 2 algorithm, Statistical support was evaluated by 1,000 bootstrap replications.

Results

Targets selection for identification of Leishmania species

According to the inclusion criteria described in “Material and methods”, 21 genes were screened out from 34 genes, which were previously reported to exhibit sequence polymorphism among Leishmania species (Additional file 1: Table S1). Further analysis indicated that the identity of these 21 genes were 88.3‒99.8% among different species and total 1,970 polymorphism sites were observed within them (Additional file 2: Table S2). Our further bioinformatics analysis were performed to select appropriate SNPs from these 1,970 polymorphism sites for Leishmania species identification. The alignment of 29 sequences of HGPRT from nine Leishmania species indicated that two SNPs can distinguish between L. donovani and L. infantum (Fig. 1). Moreover, 1‒2 SNPs were found by comparison of 43 sequences of SPDSYN from 18 Leishmania species, which can distinguish Leishmania species among L. major, L. tropica and L. donovani/L. infantum well (Fig. 2). Thus, two potential targets for Leishmania species identification, HGPRT and SPDSYN, were screened out for further investigations.

Development of allele specific real-time PCR assay for Leishmania species identification

To verify the potential application of HGPRT and SPDSYN in Leishmania species identification, the primers and probes were designed according to the conserved sequence of HGPRT and SPDSYN and the SNPs screened out above (Table 2).

Firstly, PCRs were performed with template from clinical samples or constructed plasmids. As expected, the primers, VL-HGPRT-F2 and VL-HGPRT-R1 for HGPRT and CL-SPD-F and CL-SPD-R for SPDSYN, can amplify a 145 bp fragment from L. donovani, L. infantum and L. major samples, and 202 bp fragment from L. major, L. tropica, L. donovani and L. infantum samples respectively. In addition, these two pair of primers didn’t recognize any DNA from samples of P. falciparum, T. gondii, B. melitensis and O. tsutsugamushi (Fig. 3). These results indicated that the targets we selected here were specific for Leishmania species detection, which were potentially appropriate for further allele-specific real-time PCR assay construction.

Fig. 3
figure 3

Specificity of the primers designed for HGPRT (lane 2‒9) and SPDSYN (lane 10‒18) amplification. The allele specific real time PCR assays exhibit no cross-reactions with Plasmodium falciparum, Toxoplasma gondii, Brucella melitensis and Orientia tsutsugamushi

Full size image

Then an allele specific real-time PCR assay for Leishmania species identification were established using the primers and probes described above. Our results showed that this assay can detect 3 parasites/reaction for VL by targeting at HGPRT and 12 parasites/reaction for CL with SPDSYN (Additional file 3: Table S3).

The standard curves of this assay were also obtained using serially diluted plasmid DNA. It showed the PCR efficiency with both single-species and multi-species samples reactions were similar and the amplification curve were coincident as well (Fig. 4). The linear were over a 7-log range with a correlation coefficient (R2) of 0.995‒0.999 for VL (Fig. 4A and B) and 6/7-log range with a R2 of 0.994‒0.999 for CL (Fig. 4C, D and G).

Fig. 4
figure 4

Quantitative correlation between gene copy number and threshold cycle of the allele specific real-time PCR assay. A, B HGPRT plasmid was serially diluted from 102 to 108 copies/reaction and subjected to allele specific qPCR. Linear regression of Ct vs lg copy number of HGPRT plasmid were generated using single and multi-species templates for L. donovani (A) and L. infantum (B) detection, individually. C SPDSYN plasmid was serially diluted from 102 to 108 copies/reaction and subjected to allele specific qPCR. Linear regression of Ct vs lg copy number of SPDSYN plasmid were generated using single and multi-species templates for L. major detection. D, E SPDSYN plasmid was serially diluted from 103 to 109 copies/reaction and subjected to allele specific qPCR. Linear regression of Ct vs lg copy number of SPDSYN plasmid were generated using single and multi-species templates for L. tropica (D) and L. donovani/infantum (E) detection, respectively. ΔRn = Rn (normalized reporter)-baseline. Ct Cycle threshold

Full size image

Moreover, both intra-CV% and inter-CV% of Ct values for 20 replicates were < 2% (Additional file 4: Table S4). All these results implied that this allele-specific real-time PCR assay exhibited high precision for VL and CL species identification.

Validation the established Leishmania species identification assay

As the allele-specific real-time PCR assay we developed above exhibited high PCR efficiency and precision, total 42 clinical samples were used to validate the performance of this assay (Table 3). For 22 clinical VL samples, the new method detected 3 as L. donovani infections and 19 as L. infantum, which was consistence with the sequencing results. Similarly, 20 skin lesion CL samples were all identified as L. major using by this new method and confirmed by sequencing as well.

Table 3 The allele-specific real-time PCR results with the samples from 42 patients
Full size table

A phylogenetic tree was constructed using 29 Leishmania HGPRT sequences (145 bp) from nine Leishmania species and 22 VL clinical samples. The clustering results shows that 3/22 clinical samples (patient ID 10, 11, 19) were clustered with L. donovani and 19/22 clinical samples were clustered with L. infantum (Fig. 5 and Table 3). Also, phylogenetic analysis with 43 SPDSYN gene sequences (202 bp) from18 Leishmania species and 20 CL clinical samples indicated that 20 clinical samples were all clustered with L. major (Fig. 6 and Table 3). Both of these two clustering outcomes were consistence with the new methods we developed here, which further confirmed the reliability of this new assay for Leishmania species identification.

Fig. 5
figure 5

Neighbor-Joining was used to generate phylogenetic tree with 1000 replications for bootstrap based on HGPRT gene fragment, including sequences from patients’ samples

Full size image
Fig. 6
figure 6

Neighbor-Joining was used to generate phylogenetic tree with 1000 replications for bootstrap based on SPDSYN gene fragment, including sequences from patients’ samples

Full size image

Discussion

In this study, HGPRT and SPDSYN genes, which exhibit species-specific SNPs, were selected based on the screening of 21 housekeeping gene sequences from 9 species of VL and 18 species of CL. According to the conserved regions and species-specific SNPs, primers and probes were designed to perform two allele specific real-time PCR assays respectively. Our results showed that this new developed assay could identify the Leishmania species for VL between L. donovani and L. infantum with HGPRT and for CL among L. major, L. tropica and L. donovani/L. infantum with SPDSYN.

Previous studies identified Leishmania species using a SYBR-green based qPCR followed by melting analysis. Several different target were in these assays, including ITS1 for Leishmania (Viannia) spp., L. donovani complex, L. tropica, L. mexicana, L. amazonensis, L. major, and L. aethiopica [33]; canine beta-2-microglobulin and human glyceraldehyde-3 phosphate dehydrogenase for Leishmania (Viannia) spp., L. infantum and L. amazonensis [34]; amino acid permease 3 and cytochrome oxidase II (COII) genes for L. major, L. tropica and mix infections [35]; minicircle kDNA for the subgenera Leishmania and Viannia [36]; Cyt b gene for L. braziliensis, L. guyanensis, L. infantum, L. major, L. tropica and L. panamensis [27], and glucose-6-phosphate dehydrogenase for L. braziliensis or L. peruviania from the other Leishmania (Viannia) spp [32]. Although this type of assay was simple and cost-consuming, it is less specific and the results analysis was more complicated compared to the probe-based real-time PCR [25, 37].

There are also some real-time PCR identification methods were developed with different detecting targets, such as cathepsin L-like cysteine protease B gene for L. major, L. tropica and L. aethiopica [38]; amino acid permease 3 (AAP3) and COII for L. major and L. tropica [39], and glucose phosphate isomerase (GPI) for Leishmania (Viannia) spp., L. mexicana complex, L. infantum/donovani complex and L. major complex [31]. The two allele-specific qPCR assays we developed here were focused on the Leishmania species that are common in clinical practice, such as L. donovani, L. infantum, L. major and L. tropica. Using two firstly reported targets, HGPRT and SPDSYN genes with species-specific SNPs, the LOD of these assays was 3 and 12 parasites/reaction for VL and CL, individually and no cross-reaction with P. falciparum, T. gondii, B. melitensis, O. tsutsugamushi and non-target species of Leishmania was detected (Additional file 3: Table S3; Figs. 4 and 5). Considering it takes only 2.5 h to identify Leishmania species directly from clinical samples without parasites isolation or culture, these assays are suitable in clinical practice.

A total of 42 clinical samples (22 VL and 20 CL) were used to evaluate the performance of the allele-specific real-time PCR assay, which identified 22 VL clinical samples as L. donovani (n = 3) and L. infantum (n = 19), 20 CL clinical samples as L. major (n = 20). These results were consistent with the following sequencing analysis, which indicated that these new tools can distinguish SNPs among different Leishmania species well (Table 3). Further phylogenetic analysis was performed to validate the results of these allele-specific qPCR assays, which confirmed their reliability for potential clinical applications (Figs. 5 and 6).

HGPRT gene encoded hypoxanthine phosphoribosyl transferase, which is a central enzyme in the purine recycling pathway of all protozoan parasites [40]. Spermidine synthase encoded by SPDSYN gene is a key enzyme in the polyamine biosynthetic pathway of protozoan parasites [41]. These two housekeeping gene sequences exhibit observed interspecies polymorphism, which imply that our assays in this study could be applied to distinguish not only Leishmania species we described here, but also other species not included in this study. Indeed, our phylogenetic analysis implied that the sequence of HGPRT gene could differentiate more Leishmania species than we tested here, including L. major, L. mexicana complex and Leishmania (Viannia) subgenus (Fig. 5). Meanwhile, SPDSYN gene fragment appears to be able to distinguish Leishmania (Viannia) braziliensis, L. mexicana complex and Leishmania (Viannia) subgenus as well (Fig. 6). Further investigations are worthwhile to be performed to extend the potential scope of these identification assays.

Broad variations are noted in efficacies of leishmaniasis treatment depending on the Leishmania species, which identification would be helpful in clinical practice. For example, antimonial and miltefosine are more effective to L. major and L. donovani than L. infantum [16, 42, 43]. Unlike L. major, L. tropica appears unresponsive to PM-based ointments [44, 45]. Amphotericin B is used to treat L. tropica or L. major related CL, but not L. infantum [46,47,48]. The efficacy rates of azoles for L. infantum, L. donovani, L. major and L. tropica were 88%, 80%, 53% and 15%, respectively [49]. Further, Leishmania species-specific administrations were applied for better clinical efficiency. For L. tropica infection, intralesional treatment was more efficient than intramuscular administration with sodium stibogluconate [50]. Intravenous antimonial treatment could produce better cure rates against L. panamensis or L. braziliensis related CL compared with L. major [51,52,53]. Thus, as a rapid and accurate tool for Leishmania species identification, it would be helpful for species-adapted therapeutic schedule and patient management.

Unfortunately, MLEE, the “gold standard” method for Leishmania species identification, could not be performed in this study, due to only a few of parasites can be cultured in vitro from our stored clinical samples. Instead of MLEE, phylogenetic analysis of HGPRT and SPDSYN was applied to further confirm our species distinguish results. Although our new methods with HGPRT and SPDSYN can distinguish between L. donovani and L. infantum of VL and among L. major, L. tropica and L. donovani/infantum of CL accurately, a larger sample size should be investigated in future for further confidence, especially with clinical samples of L. tropica infection and different species co-infection which were not applied in this study.

Conclusions

A novel probe-based allele-specific real-time PCR assay was established with newly reported targets, HGPRT and SPDSYN, which could identify Leishmania species between L. donovani and L. infantum for VL, and among L. major, L. tropica and L. donovani/infantum for CL. This method could be applied for not only Leishmania species-adapted therapeutic management but also ecological and epidemiological studies.

Availability of data and materials

All data generated or analysed during this study are included in this published article.

Abbreviations

VL:

Visceral leishmaniasis

CL:

Cutaneous leishmaniasis

ML:

Mucosal leishmaniasis

PKDL:

Post kala-azar dermal leishmaniasis

RFLP:

Restriction fragment length polymorphism

MLEE:

Multi-site enzyme electrophoresis

HGPRT:

Hypoxanthine-guanine phosphoribosyl transferase

SPDSYN:

Spermidine synthase

SNPs:

Single nucleotide polymorphisms

LOD:

Limit of detection

AAP3:

Amino acid permease 3

COII:

Cytochrome oxidase II

GPI:

Glucosephosphate isomerase

PM:

Paromomycin

References

  1. Akhoundi M, Kuhls K, Cannet A, Votypka J, Marty P, Delaunay P, et al. A historical overview of the classification, evolution, and dispersion of Leishmania parasites and sandflies. PLoS Negl Trop Dis. 2016;10(3):e0004349.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  2. World Health Organization. Control of the leishmaniases: report of a meeting of the WHO expert committee on the control of leishmaniases. Geneva: World Health Organization; 2010.

    Google Scholar 

  3. Burza S, Croft SL, Boelaert M. Leishmaniasis. Lancet. 2018;392(10151):951–70.

    Article  PubMed  Google Scholar 

  4. Bi K, Chen Y, Zhao S, Kuang Y, John Wu CH. Current visceral leishmaniasis research: a research review to inspire future study. Biomed Res Int. 2018;2018:9872095.

    Article  PubMed  PubMed Central  Google Scholar 

  5. de Vries HJ, Reedijk SH, Schallig HD. Cutaneous leishmaniasis: recent developments in diagnosis and management. Am J Clin Dermatol. 2015;16(2):99–109.

    Article  PubMed  PubMed Central  Google Scholar 

  6. Torres-Guerrero E, Quintanilla-Cedillo MR, Ruiz-Esmenjaud J, Arenas R. Leishmaniasis: a review. F1000Research. 2017;6:750.

    Article  PubMed  PubMed Central  Google Scholar 

  7. Van der Auwera G, Bart A, Chicharro C, Cortes S, Davidsson L, Di Muccio T, et al. Comparison of Leishmania typing results obtained from 16 European clinical laboratories in 2014. Euro Surveill. 2016;21(49):30418.

    PubMed  PubMed Central  Google Scholar 

  8. Singh S, Sharma U, Mishra J. Post-kala-azar dermal leishmaniasis: recent developments. Int J Dermatol. 2011;50(9):1099–108.

    Article  PubMed  Google Scholar 

  9. Rogers MB, Hilley JD, Dickens NJ, Wilkes J, Bates PA, Depledge DP, et al. Chromosome and gene copy number variation allow major structural change between species and strains of Leishmania. Genome Res. 2011;21(12):2129–42.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Vandeputte M, van Henten S, van Griensven J, Huits R, Van Esbroeck M, Van der Auwera G, et al. Epidemiology, clinical pattern and impact of species-specific molecular diagnosis on management of leishmaniasis in Belgium, 2010–2018: a retrospective study. Travel Med Infect Dis. 2020;38:101885.

    Article  PubMed  Google Scholar 

  11. Bifeld E, Clos J. The genetics of Leishmania virulence. Med Microbiol Immunol. 2015;204(6):619–34.

    Article  CAS  PubMed  Google Scholar 

  12. Madusanka RK, Silva H, Karunaweera ND. Treatment of cutaneous leishmaniasis and insights into species-specific responses: a narrative review. Infect Dis Ther. 2022;11(2):695–711.

    Article  PubMed  PubMed Central  Google Scholar 

  13. Machado PR, Ampuero J, Guimaraes LH, Villasboas L, Rocha AT, Schriefer A, et al. Miltefosine in the treatment of cutaneous leishmaniasis caused by Leishmania braziliensis in Brazil: a randomized and controlled trial. PLoS Negl Trop Dis. 2010;4(12):e912.

    Article  PubMed  PubMed Central  Google Scholar 

  14. Chrusciak-Talhari A, Dietze R, Chrusciak Talhari C, da Silva RM, Gadelha Yamashita EP, de Oliveira PG, et al. Randomized controlled clinical trial to access efficacy and safety of miltefosine in the treatment of cutaneous leishmaniasis caused by Leishmania (Viannia) guyanensis in Manaus, Brazil. Am J Trop Med Hyg. 2011;84(2):255–60.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Oliveira LF, Schubach AO, Martins MM, Passos SL, Oliveira RV, Marzochi MC, et al. Systematic review of the adverse effects of cutaneous leishmaniasis treatment in the New World. Acta Trop. 2011;118(2):87–96.

    Article  CAS  PubMed  Google Scholar 

  16. Monge-Maillo B, Lopez-Velez R. Therapeutic options for old world cutaneous leishmaniasis and new world cutaneous and mucocutaneous leishmaniasis. Drugs. 2013;73(17):1889–920.

    Article  CAS  PubMed  Google Scholar 

  17. Borsari C, Jimenez-Anton MD, Eick J, Bifeld E, Torrado JJ, Olias-Molero AI, et al. Discovery of a benzothiophene-flavonol halting miltefosine and antimonial drug resistance in Leishmania parasites through the application of medicinal chemistry, screening and genomics. Eur J Med Chem. 2019;183:111676.

    Article  PubMed  CAS  Google Scholar 

  18. van Griensven J, Gadisa E, Aseffa A, Hailu A, Beshah AM, Diro E. Treatment of cutaneous leishmaniasis caused by Leishmania aethiopica: a systematic review. PLoS Negl Trop Dis. 2016;10(3):e0004495.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  19. Barkati S, Ndao M, Libman M. Cutaneous leishmaniasis in the 21st century: from the laboratory to the bedside. Curr Opin Infect Dis. 2019;32(5):419–25.

    Article  CAS  PubMed  Google Scholar 

  20. Blum J, Buffet P, Visser L, Harms G, Bailey MS, Caumes E, et al. LeishMan recommendations for treatment of cutaneous and mucosal leishmaniasis in travelers, 2014. J Travel Med. 2014;21(2):116–29.

    Article  PubMed  Google Scholar 

  21. Van der Auwera G, Dujardin JC. Species typing in dermal leishmaniasis. Clin Microbiol Rev. 2015;28(2):265–94.

    Article  PubMed  PubMed Central  Google Scholar 

  22. Gonzalez U, Pinart M, Reveiz L, Rengifo-Pardo M, Tweed J, Macaya A, et al. Designing and reporting clinical trials on treatments for cutaneous leishmaniasis. Clin Infect Dis. 2010;51(4):409–19.

    Article  CAS  PubMed  Google Scholar 

  23. Lachaud L, Fernándezarévalo A, Normand A, Lami P, Nabet C, Donnadieu J, et al. Identification of Leishmania by matrix assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry using a free web-based application and a dedicated mass-spectral library. J Clin Microbiol. 2017;55(10):2924–33.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Van der Auwera G, Maes I, De Doncker S, Ravel C, Cnops L, Van Esbroeck M, et al. Heat-shock protein 70 gene sequencing for Leishmania species typing in European tropical infectious disease clinics. Euro Surveill. 2013;18(30):20543.

    PubMed  Google Scholar 

  25. Ballin NZ, Onaindia JO, Jawad H, Fernandez-Carazo R, Maquet A. High-resolution melting of multiple barcode amplicons for plant species authentication. Food Control. 2019;105:141–50.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. de Almeida ME, Steurer FJ, Koru O, Herwaldt BL, Pieniazek NJ, da Silva AJ. Identification of Leishmania spp. by molecular amplification and DNA sequencing analysis of a fragment of rRNA internal transcribed spacer 2. J Clin Microbiol. 2011;49(9):3143–9.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  27. Foulet F, Botterel F, Buffet P, Morizot G, Rivollet D, Deniau M, et al. Detection and identification of Leishmania species from clinical specimens by using a real-time PCR assay and sequencing of the cytochrome B gene. J Clin Microbiol. 2007;45(7):2110–5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Schönian G, Akuffo H, Lewin S, Maasho K, Nylén S, Pratlong F, et al. Genetic variability within the species Leishmania aethiopica does not correlate with clinical variations of cutaneous leishmaniasis. Mol Biochem Parasitol. 2000;106(2):239–48.

    Article  PubMed  Google Scholar 

  29. Van der Auwera G, Ravel C, Verweij JJ, Bart A, Schonian G, Felger I. Evaluation of four single-locus markers for Leishmania species discrimination by sequencing. J Clin Microbiol. 2014;52(4):1098–104.

    Article  PubMed  PubMed Central  Google Scholar 

  30. Galluzzi L, Ceccarelli M, Diotallevi A, Menotta M, Magnani M. Real-time PCR applications for diagnosis of leishmaniasis. Parasit Vectors. 2018;11(1):273.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  31. Wortmann G, Hochberg L, Houng HH, Sweeney C, Zapor M, Aronson N, et al. Rapid identification of Leishmania complexes by a real-time PCR assay. Am J Trop Med Hyg. 2005;73(6):999–1004.

    Article  CAS  PubMed  Google Scholar 

  32. Castilho TM, Camargo LM, McMahon-Pratt D, Shaw JJ, Floeter-Winter LM. A real-time polymerase chain reaction assay for the identification and quantification of American Leishmania species on the basis of glucose-6-phosphate dehydrogenase. Am J Trop Med Hyg. 2008;78(1):122–32.

    Article  CAS  PubMed  Google Scholar 

  33. de Almeida ME, Koru O, Steurer F, Herwaldt BL, da Silva AJ. Detection and differentiation of Leishmania spp. in clinical specimens by use of a SYBR Green-based real-time PCR assay. J Clin Microbiol. 2017;55(1):281–90.

    Article  PubMed  Google Scholar 

  34. Diotallevi A, Buffi G, Ceccarelli M, Neitzke-Abreu HC, Gnutzmann LV, da Costa Lima Junior MS, et al. Data on the differentiation among Leishmania (Viannia) spp., Leishmania (Leishmania) infantum and Leishmania (Leishmania) amazonensis in Brazilian clinical samples using real-time PCR. Data Brief. 2020;28:104914.

    Article  PubMed  Google Scholar 

  35. Ghafari SM, Fotouhi-Ardakani R, Parvizi P. Designing and developing a high-resolution melting technique for accurate identification of Leishmania species by targeting amino acid permease 3 and cytochrome oxidase II genes using real-time PCR and in silico genetic evaluation. Acta Trop. 2020;211:105626.

    Article  CAS  PubMed  Google Scholar 

  36. Diotallevi A, Buffi G, Ceccarelli M, Neitzke-Abreu HC, Gnutzmann LV, da Costa Lima MS, Jr, et al. Real-time PCR to differentiate among Leishmania (Viannia) subgenus, Leishmania (Leishmania) infantum and Leishmania (Leishmania) amazonensis: application on Brazilian clinical samples. Acta Trop. 2020;201:105178.

    Article  CAS  PubMed  Google Scholar 

  37. Cutrin JM, Olveira JG, Bandin I, Dopazo CP. Validation of real time RT-PCR applied to cell culture for diagnosis of any known genotype of viral haemorrhagic septicaemia virus. J Virol Methods. 2009;162(1–2):155–62.

    Article  CAS  PubMed  Google Scholar 

  38. Nath-Chowdhury M, Sangaralingam M, Bastien P, Ravel C, Pratlong F, Mendez J, et al. Real-time PCR using FRET technology for Old World cutaneous leishmaniasis species differentiation. Parasit Vectors. 2016;9:255.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  39. Fotouhi-Ardakani R, Ghafari SM, Ready PD, Parvizi P. Developing, modifying, and validating a TaqMan real-time PCR technique for accurate identification of Leishmania parasites causing most leishmaniasis in Iran. Front Cell Infect Microbiol. 2021;11:731595.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Ansari MY, Equbal A, Dikhit MR, Mansuri R, Rana S, Ali V, et al. Establishment of correlation between in-silico and in-vitro test analysis against Leishmania HGPRT to inhibitors. Int J Biol Macromol. 2016;83:78–96.

    Article  CAS  PubMed  Google Scholar 

  41. Mishra AK, Agnihotri P, Srivastava VK, Pratap JV. Novel protein-protein interaction between spermidine synthase and S-adenosylmethionine decarboxylase from Leishmania donovani. Biochem Biophys Res Commun. 2015;456(2):637–42.

    Article  CAS  PubMed  Google Scholar 

  42. Neal R, Allen S, McCoy N, Olliaro P, Croft S. The sensitivity of Leishmania species to aminosidine. J Antimicrob Chemother. 1995;35:577–84.

    Article  CAS  PubMed  Google Scholar 

  43. Soto J, Toledo J, Gutierrez P, Nicholls RS, Padilla J, Engel J, et al. Treatment of American cutaneous leishmaniasis with miltefosine, an oral agent. Clin Infect Dis. 2001;33:e57–61.

    Article  CAS  PubMed  Google Scholar 

  44. El-On J, Weinrauch L, Livshin R, Even-Paz Z, Jacobs GP. Topical treatment of recurrent cutaneous leishmaniasis with ointment containing paromomycin and methylbenzethonium chloride. Br Med J. 1985;291:704–5.

    Article  CAS  Google Scholar 

  45. Shani-Adir A, Kamil S, Rozenman D, Schwartz E, Ramon M, Zalman L, et al. Leishmania tropica in northern Israel: a clinical overview of an emerging focus. J Am Acad Dermatol. 2005;53(5):810–5.

    Article  PubMed  Google Scholar 

  46. Ono M, Takahashi K, Taira K, Uezato H, Takamura S, Izaki S. Cutaneous leishmaniasis in a Japanese returnee from West Africa successfully treated with liposomal amphotericin B. J Dermatol. 2011;38(11):1062–5.

    Article  CAS  PubMed  Google Scholar 

  47. Solomon M, Pavlotsky F, Leshem E, Ephros M, Trau H, Schwartz E. Liposomal amphotericin B treatment of cutaneous leishmaniasis due to Leishmania tropica. J Eur Acad Dermatol Venereol. 2011;25(8):973–7.

    Article  CAS  PubMed  Google Scholar 

  48. Hervas JA, Martin-Santiago A, Hervas D, Rojo E, Mena A, Rocamora V, et al. Old world Leishmania infantum cutaneous leishmaniasis unresponsive to liposomal amphotericin B treated with topical imiquimod. Pediatr Infect Dis J. 2012;31(1):97–100.

    Article  PubMed  Google Scholar 

  49. Galvao EL, Rabello A, Cota GF. Efficacy of azole therapy for tegumentary leishmaniasis: a systematic review and meta-analysis. PLoS ONE. 2017;12(10):e0186117.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  50. Reithinger R, Mohsen M, Wahid M, David JR, Bismullah M, Quinnell RJ, et al. Efficacy of thermotherapy to treat cutaneous leishmaniasis caused by Leishmania tropica in Kabul, Afghanistan: a randomized, controlled trial. Clin Infect Dis. 2005;40(8):1148–55.

    Article  CAS  PubMed  Google Scholar 

  51. Arana BA, Navin TR, Arana FE, Berman JD, Frank R. Efficacy of a short course (10 Days) of high-dose meglumine antimonate with or without interferon-v intreating cutaneous leishmaniasis in Guatemala. Clin Infect Dis. 1994;3:381–4.

    Article  Google Scholar 

  52. Wortmann G, Miller RS, Oster C, Jackson J, Aronson N. A Randomized, double-blind study of the efficacy of a 10- or 20-day course of sodium stibogluconate for treatment of cutaneous leishmaniasis in United States military personnel. Clin Infect Dis. 2002;35(3):261–7.

    Article  CAS  PubMed  Google Scholar 

  53. Aronson NE, Wortmann GW, Byrne WR, Howard RS, Bernstein WB, Marovich MA, et al. A randomized controlled trial of local heat therapy versus intravenous sodium stibogluconate for the treatment of cutaneous Leishmania major infection. PLoS Negl Trop Dis. 2010;4(3):e628.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  54. Wu Y, Tian X, Song N, Huang M, Wu Z, Li S, et al. Application of quantitative PCR in the diagnosis and evaluating treatment efficacy of leishmaniasis. Front Cell Infect Microbiol. 2020;10:581639.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We would like to thank Prof. Junping Peng, at the Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, for technical assistance. We thank Drs. Lei Wang and Fei Wang for providing clinical samples needed for the experiment.

Funding

This work was supported by the National Natural Science Foundation of China under Grant No. 8207080300 to GY and the Research Foundation of Friendship Hospital, Capital Medical University under Grant No. yyqdkt2019-42 to YW. The founders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author information

Authors and Affiliations

  1. Beijing Institute of Tropical Medicine, Beijing Friendship Hospital, Capital Medical University, 95 Yong’an Road, Xi Cheng District, Beijing, 100050, China

    Yun Wu, Mengyuan Jiang, Shaogang Li & Guowei Yang

  2. Warwick Medical School, Warwick University, Coventry, UK

    Nicholas R. Waterfield

Authors
  1. Yun Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mengyuan Jiang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shaogang Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Nicholas R. Waterfield
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Guowei Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

YW, GY designed the study and interpreted the findings. YW, MJ and SL contributed to data collection and validation. YW conducted data analysis and writing of this original draft. GY and NRW review and editing. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Guowei Yang.

Ethics declarations

Ethics approval and consent to participate

This project has been approved by the Ethics Committee of Beijing Friendship Hospital (Beijing, China) with approval number of 2021-P2-356-01. All clinical samples investigated in this study were obtained from an existing sample collection. All samples were anonymized.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Supplementary Information

Additional file 1: Table S1.

34 housekeeping genes of Leishmania with sequence polymorphism.

Additional file 2: Table S2.

Leishmania interspecies polymorphism in 21 genes.

Additional file 3: Table S3.

The sensitivity of allele-specific real-time PCR assay.

Additional file 4: Table S4.

Precision of intra and inter-assay of allele-specific real-time PCR assay.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Wu, Y., Jiang, M., Li, S. et al. Establish an allele-specific real-time PCR for Leishmania species identification. Infect Dis Poverty 11, 60 (2022). https://doi.org/10.1186/s40249-022-00992-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s40249-022-00992-y

Keywords

Infectious Diseases of Poverty

ISSN: 2049-9957

Contact us