Skip to main content

Concurrent molecular characterization of sand flies and Leishmania parasites by amplicon-based next-generation sequencing



Phlebotomine sand flies are vectors of Leishmania parasites, which are the causative agents of leishmaniasis. Herein, we developed an amplicon-based next-generation sequencing (Amp-NGS) to characterize sand flies and Leishmania parasites simultaneously targeting partial fragments of 18S rDNA and ITS1 genes, respectively.


Our assay was optimized using reference sand fly (n = 8) and Leishmania spp. (n = 9) samples and validated using wild-caught sand flies from Palestine. The assay was highly specific, and all DNA references were successfully identified to the species level.


Among the wild-caught sand flies (n = 187), Phlebotomus spp. represented 95% of the collected samples (177/187), including Ph. sergenti (147/187, 79%), Ph. papatasi (19/187, 10.2%), Ph. perfiliewi (3/187, 1.6%), Ph. tobbi (2/187, 1.2%) and Ph. syriacus (6/187, 3.2%). Sergentomyia spp. represented only 5% (10/187) of the collected samples and included S. dentata (n = 6), S. fallax (n = 2), S. schwetzi (n = 1) and S. ghesquiere (n = 1). The study observed strong positive correlation between sand fly identification results of the Amp-NGS and morphological identification method (r = 0.84, df = 185, P < 0.001). Some discrepancies between the two methods in the identification of closely related species (i.e. Ph. perfiliewi, Ph. tobbi and Ph. syriacus) were observed. Leishmania DNA was detected and identified as L. tropica in 14 samples (14/187, 7.5%).


Our assay was sensitive to detect (limit of detection was 0.0016 ng/reaction) and identify Leishmania DNA in sand flies, thus representing a new tool for studying sand flies and their associated Leishmania parasites in endemic areas.

Graphical Abstract


Leishmaniasis is a group of diseases that affect millions of people worldwide, causing different clinical signs, ranging from self-limiting skin lesions to death. Among Leishmania spp. described worldwide, about 20 cause disease in humans, including cutaneous leishmaniasis (CL), visceral leishmaniasis (VL) and mucocutaneous leishmaniasis (MCL) [1]. These forms of leishmaniasis may overlap in distribution. For instance, Palestine is a traditional focus of both CL and VL. In particular, cases of human VL and canine leishmaniasis (CanL) caused by Leishmania infantum are sporadically reported, whereas CL caused by Leishmania tropica and Leishmania major is distributed all over the Palestinian districts except in Gaza Strip, which is free from the disease [2,3,4,5,6].

Leishmania parasites are transmitted through the bite of infected phlebotomine sand flies (Diptera: Psychodidae). Thus, studying Leishmania parasites and their associated sand fly vectors in endemic areas is pivotal in leishmaniasis control, as well as in understanding the transmission dynamics of these parasites in high-risk areas [7,8,9,10,11].

Several phlebotomine sand flies are regarded as vectors for Leishmania parasites [12,13,14,15,16,17,18,19,20,21,22,23], including Phlebotomus neglectus (vector of L. infantum) [12, 13], Phlebotomus sergenti (vector of L. tropica) and Phlebotomus papatasi (vector of L. major) [14]. Besides, the detection of L. infantum and L. major DNA in Sergentomyia spp. may suggest that these species could acquire the parasites while feeding on infected hosts such as rodents [15,16,17,18]. However, their vector competences and ability to become infected and to transmit these parasites to other naïve hosts have not been proven yet [15,16,17,18].

Palestine is a classical focus of CL and VL. Cases of human VL (hVL) and canine leishmaniasis (canL) caused by Leishmania infantum are sporadically reported. Cases of CL caused by L. tropica and L. major are distributed all over the Palestinian districts except in Gaza Strip, which is free from the disease [7,8,9,10,11]. Several sand fly species are regarded as proven vectors for Leishmania parasites such as Phlebotomus neglectus, a proven vector of L. infantum [12, 13]; Phlebotomus sergenti, a vector of L. tropica; and Ph. papatasi, a vector of L. major [14]. Besides, detection of L. infantum and L. major DNA in Sergentomyia species may suggest that these species could acquire the parasites while feeding on infected hosts such as rodents [15]. However, their vector competences and ability to become infected and to transmit these parasites to other naïve hosts have not been proven yet [15,16,17,18]. Sand flies of the genus Sergentomyia are present throughout the Middle East including Palestine, but without any signs of vector competence [19, 20] such as in Greece and Cyprus [21,22,23].

Phlebotomine sand fly species identification is traditionally based on microscopic examination of key morphological features [7,8,9,10,11]. This method is time-consuming and requires specific skills for specimen preparation and identification [24]. Molecular techniques targeting several ribosomal, mitochondrial and nuclear DNA have been increasingly used for the identification of sand fly species [24,25,26,27,28,29,30]. In a similar manner, several targets have been used for Leishmania parasite detection and identification in sand flies, including the kinetoplast DNA (kDNA), internal transcribed spacer 1 (ITS1) region [31,32,33], and heat shock protein 70 (hsp70) [34]. PCR assays employing these targets present variable degrees of sensitivity and specificity. For instance, most kDNA-based PCR usually present high sensitivity but low specificity. In this regard, ITS1-based PCR-RFLP (PCR-restriction fragment length polymorphism) has been used for detection and identification of Old World Leishmania spp. [31,32,33,34].

Next-generation sequencing (NGS) technologies have several advantages over classical molecular-based methods, including the ability to generate large quantities of DNA sequences in a single run for in-depth detection of pooled DNA in several DNA samples [35]. High-throughput screening makes DNA sequencing cheaper, faster and more reliable. Recently, Abbasi et al. [36] used this technology for Leishmania and sand fly identification, using ITS1 gene and cytochrome oxidase I, respectively. In the present study, we developed a novel amplicon-based NGS (Amp-NGS) method for the identification of Phlebotomus and Sergentomyia species with concomitant detection and identification of Leishmania spp. in these sand flies.


Sand fly collection and DNA extraction

Sand fly collections were made between May and September 2019 using CDC light traps (John W. Hock USA). Two traps per den for three dens were placed in two regions (Al-Khalil and Tubas) where L. major, L. tropica and L. infantum have been reported [8, 37]. All traps were left in the field from dusk to dawn for 3 consecutive nights (36 h). Sand flies were sorted using a Discovery V12 stereomicroscope (Zeiss, Germany). Female sand flies were separated and identified using the taxonomic keys [38,39,40,41]. All samples were preserved individually in 1.5-ml micro-tubes containing 70% ethanol. The female collection was subjected to DNA extraction while male samples were kept frozen for future study. Genomic DNA was extracted using a phenol-chloroform method [42]. The DNA was eluted in 50 µl of 1× TE buffer (Tris 10 mM, 1 mM EDTA, pH ~ 8) and quantified by NanoDrop 1000 (Thermo Scientific, USA) and stored at 4 °C until use.

Reference DNA samples

DNA samples from sand flies of the genera Phlebotomus (i.e. Ph. sergenti, Ph. perfiliewi, Ph. syriacus, Ph. papatasi, Ph. tobbi, Ph. argentipes), Sergentomyia (S. schwetzi) and Lutzomyia (L. guyanensis) were used as DNA standards. The samples were kindly provided by the Prof. Alon Warburg, entomology laboratory (Jerusalem). The samples were identified by microscopic examination and/or by PCR targeting the cytochrome oxidase subunit I (COI) gene The GenBank accession numbers are: Ph. papatasi (JX105037), Ph. sergenti (JX105039.1), Ph. argentipes (JX105038.1). Moreover, DNA samples from different Leishmania spp. (three samples of L. tropica, two L. major, two L. infantum, one L. donovani and one L. aethiopica) were also used in this study (Additional file 1: Table S1).

Target genes, primers and probes

To design primers for sand fly identification, representative 18S rDNA sequences (n = 29) were retrieved from GenBank, used in FASTA format and aligned using the multiple sequence alignment online program ( [43] to identify the conserved and polymorphic regions. At least two DNA sequences from the same species but with different accession numbers were included to check the stability of the polymorphic regions among the species. A conserved region in the 18S rDNA was selected to design primers able to detect different sand fly genera and species. Within the selected 18S rDNA region, a polymorphic 150 bp DNA sequence was chosen to differentiate sand flies to the species level. The forward primer was used as described elsewhere [44] while the reverse primer was selected using primer3 ( (Fig. 1). Sand fly 18S rDNA sequences used in this study and their GenBank accession numbers are shown in Additional file 1: Table S2. Likewise, the ITS1 region (~ 300 bp) was chosen for Leishmania spp. detection and identification as described previously [33]. In brief, 13 ITS1 sequences were retrieved from the GenBank (Additional file 1: Table S2), representing different geographic regions to target the conserved species-specific sites (Fig. 2). All primers were modified by adding the Illumina overhang adapter sequences at the 5’ end to fit with Illumina platform NGS system. Primer names, sequences and the expected size of the amplified products are shown in Table 1. Polymorphic sequences in both targets (18S rDNA and ITS1) were used to create virtual specific probes that can be used in our workflow bioinformatics analysis to identify the sand fly and Leishmania species using the Galaxy, a free online program ( Probes are shown in Additional file 1: Table S3.

Fig. 1
figure 1

Multiple sequence alignment of the 18S rDNA nucleotide sequences of several sand fly species for designing reverse primer and species-specific probes. Red fonts represent the identical sequences, while blue and black show the differences between the sand fly species that were used for virtual probe selection to identify the sand fly species

Fig. 2
figure 2

Multiple sequence alignment of the ITS1 nucleotide sequences of several Leishmania species to design species-specific probes as indicated in the green boxes. L. tropica 1, 2 (Israeli), 3 from Iran, L. aethiopica from Ethiopia, L. major 1 from Ashkhabad (5ASKH), L. major 2 from Iraq, L. infantum 1 and 2 from Tunisia and France, respectively, L. donovani 1 and 2 from India and Sudan, respectively

Table 1 Primer sequences and corresponding target genes used in the study

Multiplex PCR for detection of Leishmania and sand fly DNA

For simultaneous detection and identification of sand fly and Leishmania DNA, two primer sets were used for multiplex PCR, ITS1NGSF/ITS1NGSR and SFNGSF/SFNGSR, which amplified a fragment of 343 bp of Leishmania spp. ITS1 and a fragment of 230 bp of sand fly 18S rRNA, respectively. For standardization of our multiplex PCR, different concentrations of SFNGSF and SFNGSR primers (1.0, 0.1, 0.01 and 0.001 µM) were used in the reaction mixture to verify the lowest concentration of primers needed for amplifying the sand fly 18S rDNA fragment. The lowest detection limit of Leishmania DNA was determined using tenfold serial dilutions of pure L. tropica DNA (1–0.00032 ng), which were added to 10 ng of Ph. sergenti DNA and subjected to multiplex PCR. Following optimization, the PCR reaction mixture composed of 12.5 μl of PrimeSTAR Max Premix (2×) (Takara Bio Inc., JP), 1 μM of ITS1NGSF and ITS1NGSR primers, and 0.1 μM of SFNGSF and SFNGSR primers and 2.5 μl of DNA template for a final volume of 25 μl. To validate our assay, 17 DNA reference samples (eight sand fly DNA samples and nine Leishmania DNA samples) were tested (Additional file 1: Table S1), and then all field-collected female sand flies were subjected to multiplex PCR for simultaneous detection and identification of sand flies and Leishmania parasites.

DNA library preparation

The multiplex PCR products (25 μl) were cleaned by AMPure XP beads, Beckman Coulter (X1), and eluted in 25 μl elution buffer. The purified products (7.5 μl) were subjected to a second PCR to include unique index sequences (N7XX and S5XX) for barcoding of each sample using Nextera XT Index Kit (Illumina, San Diego, CA, USA). Then, 5 μl from each barcoded sample was pooled together, mixed and spun down. Finally, 100 μl of pooled DNA was purified using X1 AMPure XP beads and quantified by Qubit® Fluorometer (Invitrogen) machine. The concentration of 4.0 nM was prepared from the pooled sample. At least 10,000 reads for each sample were targeted. Samples were deep-sequenced with the Nextseq500 machine using the 150-cycle mid output kit (Illumina, Inc., USA) from the forward read direction.

Bioinformatics analysis

Binary Base Call (BCL) output files from a Nextseq 500 machine were converted to FASTQ format, using BCL to FASTQ (bcl2fastq v2.20.0.422 Copyright (c) 2007–2017 Illumina, Inc.). The files were analyzed using the Galaxy program (Galaxy Version ( Initially, the obtained sequences were run through fastqc ( to check the quality of the generated reads. The sequences were then trimmed ( using default parameters to retain the highest quality reads (reads above 100 bp) and a minimum quality score of > 20, which represents an error rate of 1 in 100 (according to Illumina Nextseq machine sequencing error rate), with a corresponding call accuracy of 99%. The filtered data were captured by virtual specific sand fly and Leishmania probes at the genus and species levels. Based on the species-specific probes, the number of sequence reads for each species was determined. When non-Leishmania or non-sand fly species were detected, the dominant sequence was megablasted ( to confirm the species. The cutoff value of 200 reads per sample was set up to determine Leishmania positivity as described previously [45].

Statistical analysis and phylogenetic tree

Statistical analysis was performed using Statistical Package for the Social Sciences (SPSS) version 20.0 (IBM Corp.) to test the Spearman’s correlation to measure the degree of correlation between classical microscopically results and the Amp-NGS results. GraphPad free online program ( was used to calculate the degree of agreement (kappa). The free online program was used for kappa agreement tests. Cohen’s kappa coefficient (k) is a measure of the agreement between two tests beyond that expected by chance, where 0 is chance agreement and 1 is perfect agreement for the measure the degree of association between classical microscopically results and performance against the Amp-NGS results assay. The significance tests were two-tailed. The differences were considered statistically significant when P-values were < 0.05.

Phylogenetic tree construction was carried out using the statistical method maximum likelihood (ML) with a bootstrap value of 1000 replications using MEGA X program [46]. Partial DNA sequences of 18S rDNA (150 bp) and ITS1 (150 bp) were used to build the phylogenetic tree based on complete deletion option with gaps and missing data were eliminated. These two 150-bp sequences were produced by the Illumina nextseq500 sequencing machine using the 150-cycle mid output kit (Illumina, Inc., USA). All were based on the Jukes‐Cantor model for nucleotide sequences. Initial tree for the heuristic search was automatically obtained by applying the nearest‐neighbor‐interchange (NNI) algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood (MCL) approach.


Morphological identification of sand flies

A total of 187 female sand flies were collected from Tubas, northern Palestine, a focus of CL caused by L. tropica, and from Bet Oula, Al-Khalil, southern Palestine (endemic for VL caused by L. infantum). All sand flies were identified morphologically as belonging to nine species (Table 2). The most abundant species were Ph. sergenti (n = 143, 76.5%) and Ph. papatasi (n = 21, 11.2%), representing together 87.7% of the sand flies collected.

Table 2 Microscopic identification of field-collected sand flies from Tubas and Bet Oula, 2018

Sensitivity and specificity of the multiplex PCR assay

Before optimizing the multiplex PCR assay, two singleplex PCRs were performed to detect the sand fly and Leishmania DNA utilizing reference DNA samples. The two sets of primers specific for sand flies (SFNGSF/SFNGSR) and Leishmania (ITS1NGSF/ITS1NGSR) amplified the expected targets without showing any signs of non-specific bands (data not shown). After testing different concentrations of the sand fly 18S rDNA primers (SFNGSF/SFNGSR) and the Leishmania ITS1 primers (ITS1219NGSF/ITS1219NGSR) for the best optimal primer concentration which allowed the amplification of 0.4 ng of L. tropica, DNA was 0.1 µM and 1 µM, respectively. (Fig. 3).

Fig. 3
figure 3

A 1.5% agarose gel obtained from the amplification of partial fragments of sand fly 18srDNA and Leishmania ITS1 genes with the multiplex PCR assay using different concentrations of 18SrRDNA sand fly primers (1–4: 1, 0.1, 0.01 and 0.001 µM) and 1 µM of ITS1 primers

The analytical sensitivity of the Amp-NGS assay for Leishmania DNA was 0.0016 ng/µl corresponded to 493 reads of ITS1 Leishmanial DNA sequences obtained by the Nextseq500 sequencing machine at a constant concentration of 10 ng of Ph. sergenti DNA, which corresponded to 192,389 reads of sand fly DNA sequence of 18S rDNA (Table 3). The optimized multiplex PCR was then applied to 187 field-collected sand flies producing the expected amplicon size of ~ 230 bp. Due to Illumina technology using the 150-cycle the Nextseq500 machine and mid output kit (Illumina, Inc., USA), only 150 bp was sequenced from the forward read direction.

Table 3 Serial concentrations of Leishmania DNA with their concomitant number of reads for sand flies and Leishmania parasites

As shown in Fig. 4, the lower line of bands from 1 to 18 at the size of 230 bp represented amplified DNA identical to sand fly DNA, while the upper line of bands at the size of 343 bp represented amplified DNA identical to Leishmanial DNA, which is in this case lanes 9 and 17 only.

Fig. 4
figure 4

A 1.5% agarose gel obtained from the amplification of 18 field-collected sand flies targeting the sand fly 18S rDNA and Leishmania ITS1 with multiplex PCR assay. Concentrations of 0.1 µM of 18S rDNA and 1 µM of ITS1 primers were used. MW is a 100-bp marker

Sand flies and Leishmania parasites detection by Amp-NGS

To validate Amp-NGS for identification of Leishmania and sand fly DNA, DNA from reference samples (sand flies and Leishmania) was mixed in a 1:1 volume ratio and subjected to multiplex PCR. A DNA library was prepared, cleaned and sent for deep sequencing as described above. Based on our workflow analysis, the used virtual probes specific for Leishmania (Additional file 1: Table S3) correctly identified all Leishmania reference DNA samples to the species level. Similarly, reference sand fly DNA samples (Additional file 1: Table S1) were correctly identified to the species level using species-specific virtual probes, which included the species of Ph. sergenti, Ph. perfiliewi, Ph. syriacus, Ph. papatasi, Ph. tobbi, Ph. argentipes, L. guyanensis and S. schwetzi. To assess the practical usability of Amp-NGS, the assay was applied on field-collected sand flies. Out of 187 tested sand flies, 177 (95%) belonged to Phlebotomus spp. including the two major species, Ph. sergenti (n = 147, 78.6%) and Ph. papatasi (n = 19, 10.2%), whereas only 5% (10/187) belonged to Sergentomyia spp. including S. dentata (n = 6), S. fallax (n = 2), S. ghesquiere (n = 1) and S. schwetzi (n = 1) (Table 4). The study observed strong positive Spearman’s correlation between sand fly identification results of the Amp-NGS and morphological identification method (r = 0.84, df = 185, P < 0.001).

Table 4 Comparison between the Amp-NGS method and the morphological method for identification of sand fly species

Concordance between tests was determined using the kappa index (k), which was 0.976 (kappa between 0.81 and 1.00 indicates perfect agreement) with standard error (SE) of 0.028 (95% CI 0.906 to 1.000). The two methods were in perfect agreement in 173 sand flies, but discrepant in 14 sand flies (Table 4). All Ph. sergenti (n = 143) and 19 Ph. papatasi were correctly identified by both methods with complete agreement. However, two Ph. sergenti revealed by NGS were identified as Ph. papatasi by microscopic examination. Further discrepancies in the identification of Ph. perfiliewi, Ph. tobbi, Ph. syriacus, and S. fallax were found (Table 4). Leishmania DNA was detected and identified as L. tropica in 14 samples (14/187, 7.5%). All L. tropica-positive sand flies were Ph. sergenti collected from Tubas.

To confirm the NGS sequencing, Sanger sequencing was performed for two full ITS1 regions and showed similar results to those obtained by Amp-NGS assay.

Phylogenetic analysis

Sand fly 18S rDNA sequences (150 bp) obtained from Amp-NGS assay were used to study the phylogenetic relationships between 187 field-collected sand flies included in this study and 31 sequences retrieved from the GenBank representing different species of sand flies obtained from different geographic regions. Nine DNA sequences representing the sand fly controls used in the study were also included in the phylogenetic analysis. The phylogenetic tree (n = 227) demonstrated three clearly separated clusters, Phlebotomus and Sergentomyia, the Old World sand flies, and Lutzomyia, the New World sand flies (Fig. 5). The phylogenetic analysis showed that the geographical origin did not have any effect on the clustering of the 18S rDNA sequences (Fig. 5). It was notable that the nucleotide 18S rDNA sequences of the four S. dentata identified in this study were identical to each other and to the respective S. dentata reference sequence (accession no. AJ244423.1) obtained from Greece. Furthermore, the 18S rDNA sequences of Ph. sergenti (n = 147) and Ph. papatasi (n = 19), CL vectors, formed two related sub-clusters within the Phlebotomus clade, while VL vectors, Ph. neglectus, Ph. perfiliewi, Ph. tobbi and Ph. syriacus were closely related to Ph. argentipes, another VL vector (Fig. 5).

Fig. 5
figure 5

Neighbor-joining (NJ) tree showing the relationships of the study and reference sandflies (n = 227) shown in bold red based on 150 bp of the 18srDNA gene sequences. MEGA X program was used for constructing the phylogenetic trees. DNA sequences were aligned using Clustal-W program. Bootstrap values are based on 1000 replicates [46]. Ph., Phlebotomus; S. Sergentomyia; L., Lutzomyia; Gen_AJ244374.1, GenBank accession number; Ref_sf6B, reference strain_sand fly number 6B; CL, cutaneous leishmaniasis; VL, visceral leishmaniasis; PS, Palestine with Palestinian sand fly code; numbers in parentheses indicate number of samples with identical genetic characters


Herein, we used a unique amplicon-based multiplex NGS method to concurrently identify species of wild-caught sand flies and detect naturally infecting Leishmania species. The uniqueness of this method, to our knowledge, comes from use of a one-step reaction to identify the species across both sand fly and Leishmania genera from a single sand fly. To date, most conventional molecular biology approaches were developed to identify Leishmania parasites in hosts or vectors by targeting different genes [23, 25,26,27, 32, 47,48,49,50,51,52]. The limit of detection (LoD) of these methods ranged from three promastigotes to less than one promastigote in the tested specimen [22, 23, 25,26,27, 32]. PCR-based methodologies have been developed further into multiplex PCR followed by either restriction fragment length polymorphism(RFLP) or dot blot hybridization for further identification [31, 44, 53,54,55]. The Amp-NGS in this study was able to detect Leishmania spp. and identity sand fly from a single midgut without needing any extra confirmatory or genotyping methods.

Recently, real-time PCR (qPCR) was used to identify Leishmania spp. in sand fly employing kDNA, spliced-leader RNA or vacuolar ATPase subunit C (VATP) with a LoD of 1 fg of DNA, 10–3 of a parasite and 0.162 parasite, respectively [33, 56,57,58,59,60]. The Amp-NGS was successfully able to detect both Leishmania and sand fly at a minimum concentration of 0.0016 ng (< 1 parasite) (Table 3) without any cross-hybridization between the two primer pairs and any mispriming with the other template [61]. Another study used high-resolution melt (HRM) qPCR to detect Leishmania [33]. Hitherto, only a handful of studies used NGS technology in the identification of Leishmania that used ITS1 and HSP70 (heath shock protein) with the former using longer targets (500 bp) and ignoring validity checks, thus reducing sensitivity and specificity [36, 62, 63].

In this study, multiplex Amp-NGS method was proven to be highly Leishmania-specific by correctly identifying the five Leishmania spp. controls from France, Ethiopia, Italy, India, Palestine and Iraq. At the same time, the method was highly sand fly-specific by correctly distinguishing the three genera of sand flies, Phlebotomus, Sergentomyia and Lutzomyia, as well as identifying the six Phlebotomus species controls.

Amp-NGS for both Leishmania and sand fly analyses depends on the concept of sequencing hundreds to thousands of the same amplified DNA molecules (depth) followed by generating a collapsed DNA sequence out of multiple sequences (reads) into one reliable sequence that is compared to a known reference sequence. The bioinformatics analysis of these hundreds to thousands of sequences (reads) of the same target will suppress sequence errors and performs deduplications to produce a very specific and accurate collapsed sequence. In addition, an incorrect base call probability of 1 in 100 produced sequences [lower base call accuracy of 99% (Q20)] with read length < 100 bp were removed from the analysis [64]. Moreover, our study used common and species-specific virtual probes in the polymorphic regions of the targeted sequences that ensured the validity (accuracy) and reliability (repeatability and consistency) of sand fly and Leishmania parasite identification.

The sand fly species identified by Amp-NGS were in perfect agreement with the morphological identification as the reference method [kappa index (k) = 0.976], indicating high reliability of the Amp-NGS (Table 4). The six discrepant samples were identified as Ph. syriacus by Amp-NGS, while by morphological examination, three of them were Ph. perfeliewi and the other three were Ph. tobbi. Both species belong to the subgenus Larroussius, which are morphologically hardly distinguishable (personnel communication). Similarly, some samples of Sergentomyia fallax, S. dentata and S. theodori species were interchangeably identified by the morphological method. The separation of these species is mainly based on differences in shape and armature of the structures of mouthparts and the pharynx. It is reported that some sand flies belonging to the same genus do not comply with all known morphological features, leading to misidentifications [65, 66]. The few discrepant cases between morphological and AMP-NGS could be due to missing a few recently written keys or drawings in morphological identification that could lead to the misidentification of species.

NGS is superior to Sanger sequencing in several aspects. NGS is significantly quicker, lower DNA concentration is required, and it is more accurate and reliable compared to Sanger sequencing. Furthermore, NGS has higher throughput with lower cost per sample. Unlike Sanger sequencing, NGS is able to identify contaminated DNA of more than one target in a single reaction.

Using phylogenetic analysis the Amp-NGS efficiently grouped sand flies into clusters matching the scientifically known sand fly taxonomic units on the levels of genera and species. All GenBank-retrieved and study sand flies clustered into three main genera (Phlebotomus, Sergentomiya and Lutzomyia). The genus Phlebotomus was further divided into two sub-clusters, CL and VL (Fig. 5). The congruence between the sand fly species detection by Amp-NGS and morphological identification on one side and the agreement between Amp-NGS and phylogenetic analysis on the other confirms the reliability of Amp-NGS as an identification method.

Here, despite a relatively small size of samples tested (n = 187), the main dominant genus of the sand fly in Palestine was Phlebotomus spp. (95%) compared to the low prevalent Sergentomyia (5%). Phlebotomus spp. are the main incriminated vectors of Leishmania parasite [41]. Out of the 95% Phlebotomus spp., 83% were Ph. sergenti, the L. tropica vector, and 11% were Ph. papatasi, the L. major vector, which concords with a previous study [8].


Our newly developed Amp-NGS assay was optimized and validated as a powerful tool for the simultaneous identification of sand flies and Leishmania species. This assay allows the identification of large numbers of sand flies, which may be useful for large-scale field studies.

Availability of data and materials

Representative sand fly DNA sequences were deposited in the GenBank under the accession numbers MN538937-MN538944. Two L. tropica sequences of about 270 bp detected in two sand flies were also deposited in the GenBank (accession numbers: MT966013-MT966014).



Cutaneous leishmaniasis


Visceral leishmaniasis


Amplicon-based next-generation sequencing


Mucocutaneous leishmaniasis


Human visceral leishmaniasis


Canine leishmaniasis


Internal transcribed spacer 1


Heat shock protein 70


Limit of detection


High-resolution melt


Restriction fragment length polymorphism




  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 sand flies. PLoS Negl Trop Dis. 2016;10:e0004349.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  2. Abdeen ZA, Sawalha SS, Eisenberger CL, Khanfar HM, Greenblatt CL, Yousef O, et al. Epidemiology of visceral leishmaniasis in the Jenin district, West Bank: 1989–1998. Am J Trop Med Hyg. 2002;66:329–33.

    Article  PubMed  Google Scholar 

  3. Al-Jawabreh A, Dumaidi K, Ereqat S, Al-Jawabreh H, Nasereddin A, Azmi K, et al. Molecular epidemiology of human cutaneous leishmaniasis in Jericho and its vicinity in Palestine from 1994 to 2015. Infect Genet Evol. 2017;50:95–101.

    Article  CAS  PubMed  Google Scholar 

  4. Al-Jawabreh A, Schnur LF, Nasereddin A, Schwenkenbecher JM, Abdeen Z, Barghuthy F, et al. The recent emergence of Leishmania tropica in Jericho (A’riha) and its environs, a classical focus of L. major. Trop Med Int Health. 2004;9:812–6.

    Article  CAS  PubMed  Google Scholar 

  5. Hamarsheh O, Nasereddin A, Damaj S, Sawalha S, Al-Jawabreh H, Azmi K, et al. Serological and molecular survey of Leishmania parasites in apparently healthy dogs in the West Bank. Palestine Parasit Vectors. 2012;5:183.

    Article  PubMed  Google Scholar 

  6. Amro A, Azmi K, Schonian G, Nasereddin A, Alsharabati MB, Sawalha S, et al. Epidemiology of paediatric visceral leishmaniasis in Hebron district, Palestine. Trans R Soc Trop Med Hyg. 2009;103:731–6.

    Article  PubMed  Google Scholar 

  7. Killick-Kendrick R. Phlebotomine vectors of the leishmaniases: a review. Med Vet Entomol. 1990;4:1–24.

    Article  CAS  PubMed  Google Scholar 

  8. Ready PD. Biology of phlebotomine sand flies as vectors of disease agents. Annu Rev Entomol. 2013;58:227–50.

    Article  CAS  PubMed  Google Scholar 

  9. Killick-Kendrick R. Some epidemiological consequences of the evolutionary fit between Leishmaniae and their phlebotomine vectors. Bull Soc Pathol Exot Filiales. 1985;78:747–55.

    CAS  PubMed  Google Scholar 

  10. Rigg CA, Calzada JE, Saldana A, Perea M, Chaves LF, Valderrama A. Leishmania spp. infection rate and feeding patterns of sand flies (Diptera: Psychodidae) from a hyperendemic cutaneous leishmaniasis community in Panama. Am J Trop Med Hyg. 2019;100:798–807.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Gonzalez E, Jimenez M, Hernandez S, Martin-Martin I, Molina R. Phlebotomine sand fly survey in the focus of leishmaniasis in Madrid, Spain (2012–2014): seasonal dynamics, Leishmania infantum infection rates and blood meal preferences. Parasit Vectors. 2017;10:368.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. Alten B, Maia C, Afonso MO, Campino L, Jimenez M, Gonzalez E, et al. Seasonal dynamics of phlebotomine sand fly species proven vectors of Mediterranean Leishmaniasis caused by Leishmania infantum. PLoS Negl Trop Dis. 2016;10:e0004458.

    Article  PubMed  PubMed Central  Google Scholar 

  13. Leger N, Gramiccia M, Gradoni L, Madulo-Leblond G, Pesson B, Ferte H, et al. Isolation and typing of Leishmania infantum from Phlebotomus neglectus on the island of Corfu, Greece. Trans R Soc Trop Med Hyg. 1988;82:419–20.

    Article  CAS  PubMed  Google Scholar 

  14. Maroli M, Feliciangeli MD, Bichaud L, Charrel RN, Gradoni L. Phlebotomine sand flies and the spreading of leishmaniases and other diseases of public health concern. Med Vet Entomol. 2013;27:123–47.

    Article  CAS  PubMed  Google Scholar 

  15. Jaouadi K, Ghawar W, Salem S, Gharbi M, Bettaieb J, Yazidi R, et al. First report of naturally infected Sergentomyia minuta with Leishmania major in Tunisia. Parasit Vectors. 2015;8:649.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  16. Pereira S, Pita-Pereira D, Araujo-Pereira T, Britto C, Costa-Rego T, Ferrolho J, et al. First molecular detection of Leishmania infantum in Sergentomyia minuta (Diptera, Psychodidae) in Alentejo, southern Portugal. Acta Trop. 2017;174:45–8.

    Article  CAS  PubMed  Google Scholar 

  17. Ozbel Y, Karakus M, Arserim SK, Kalkan SO, Toz S. Molecular detection and identification of Leishmania spp. in naturally infected Phlebotomus tobbi and Sergentomyia dentata in a focus of human and canine leishmaniasis in western Turkey. Acta Trop. 2016;155:89–94.

    Article  CAS  PubMed  Google Scholar 

  18. Jaouadi K, Haouas N, Chaara D, Boudabous R, Gorcii M, Kidar A, et al. Phlebotomine (Diptera, Psychodidae) bloodmeal sources in Tunisian cutaneous leishmaniasis foci: could Sergentomyia minuta, which is not an exclusive herpetophilic species, be implicated in the transmission of pathogens? Ann Entomol Soc Am. 2013;106:79–85.

    Article  Google Scholar 

  19. Sawalha SS, Shtayeh MS, Khanfar HM, Warburg A, Abdeen ZA. Phlebotomine sand flies (Diptera: Psychodidae) of the Palestinian West Bank: potential vectors of leishmaniasis. J Med Entomol. 2003;40:321–8.

    Article  PubMed  Google Scholar 

  20. Sawalha SS, Ramlawi A, Sansur RM, Salem IM, Amr ZS. Diversity, ecology, and seasonality of sand flies (Diptera: Psychodidae) of the Jenin District (Palestinian Territories). J Vector Ecol. 2017;42:120–9.

    Article  PubMed  Google Scholar 

  21. Tsirigotakis N, Pavlou C, Christodoulou V, Dokianakis E, Kourouniotis C, Alten B, et al. Phlebotomine sand flies (Diptera: Psychodidae) in the Greek Aegean Islands: ecological approaches. Parasit Vectors. 2018;11:97.

    Article  PubMed  PubMed Central  Google Scholar 

  22. Dokianakis E, Tsirigotakis N, Christodoulou V, Poulakakis N, Antoniou M. Identification of wild-caught phlebotomine sand flies from Crete and Cyprus using DNA barcoding. Parasit Vectors. 2018;11:94.

    Article  PubMed  PubMed Central  Google Scholar 

  23. Dokianakis E, Tsirigotakis N, Christodoulou V, Poulakakis N, Antoniou M. DNA sequencing confirms PCR-RFLP identification of wild caught Larroussius sand flies from Crete and Cyprus. Acta Trop. 2016;164:314–20.

    Article  CAS  PubMed  Google Scholar 

  24. Contreras Gutierrez MA, Vivero RJ, Velez ID, Porter CH, Uribe S. DNA barcoding for the identification of sand fly species (Diptera, Psychodidae, Phlebotominae) in Colombia. PLoS ONE. 2014;9:e85496.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  25. Aransay AM, Scoulica E, Chaniotis B, Tselentis Y. Typing of sand flies from Greece and Cyprus by DNA polymorphism of 18S rRNA gene. Insect Mol Biol. 1999;8:179–84.

    Article  CAS  PubMed  Google Scholar 

  26. Aransay AM, Scoulica E, Tselentis Y. Detection and identification of Leishmania DNA within naturally infected sand flies by seminested PCR on minicircle kinetoplastic DNA. Appl Environ Microbiol. 2000;66:1933–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Aransay AM, Scoulica E, Tselentis Y, Ready PD. Phylogenetic relationships of phlebotomine sand flies inferred from small subunit nuclear ribosomal DNA. Insect Mol Biol. 2000;9:157–68.

    Article  CAS  PubMed  Google Scholar 

  28. Beati L, Caceres AG, Lee JA, Munstermann LE. Systematic relationships among Lutzomyia sand flies (Diptera: Psychodidae) of Peru and Colombia based on the analysis of 12S and 28S ribosomal DNA sequences. Int J Parasitol. 2004;34:225–34.

    Article  CAS  PubMed  Google Scholar 

  29. Depaquit J. Molecular systematics applied to Phlebotomine sand flies: review and perspectives. Infect Genet Evol. 2014;28:744–56.

    Article  PubMed  Google Scholar 

  30. Depaquit J, Ferte H, Leger N, Killick-Kendrick R, Rioux JA, Killick-Kendrick M, et al. Molecular systematics of the phlebotomine sand flies of the subgenus Paraphlebotomus (diptera, psychodidae, phlebotomus) based on ITS2 rDNA sequences. Hypotheses of dispersion and speciation. Insect Mol Biol. 2000;9:293–300.

    Article  CAS  PubMed  Google Scholar 

  31. Bensoussan E, Nasereddin A, Jonas F, Schnur LF, Jaffe CL. Comparison of PCR assays for diagnosis of cutaneous leishmaniasis. J Clin Microbiol. 2006;44:1435–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Schonian G, Nasereddin A, Dinse N, Schweynoch C, Schallig HD, Presber W, et al. PCR diagnosis and characterization of Leishmania in local and imported clinical samples. Diagn Microbiol Infect Dis. 2003;47:349–58.

    Article  CAS  PubMed  Google Scholar 

  33. Talmi-Frank D, Nasereddin A, Schnur LF, Schonian G, Toz SO, Jaffe CL, et al. Detection and identification of old world Leishmania by high resolution melt analysis. PLoS Negl Trop Dis. 2010;4:e581.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  34. Montalvo AM, Fraga J, Tirado D, Blandon G, Alba A, Van der Auwera G, et al. Detection and identification of Leishmania spp.: application of two hsp70-based PCR-RFLP protocols to clinical samples from the New World. Parasitol Res. 2017;116:1843–8.

    Article  PubMed  Google Scholar 

  35. Cruaud P, Rasplus JY, Rodriguez LJ, Cruaud A. High-throughput sequencing of multiple amplicons for barcoding and integrative taxonomy. Sci Rep. 2017;7:41948.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Abbasi I, Nasereddin A, Warburg A. Development of a next generation DNA sequencing-based multi detection assay for detecting and identifying Leishmania parasites, blood sources, plant meals and intestinal microbiome in phlebotomine sand flies. Acta Trop. 2019;199:105101.

    Article  CAS  PubMed  Google Scholar 

  37. Al-Jawabreh A, Ereqat S, Dumaidi K, Nasereddin A, Sawalha S, Al-Jawabreh H, et al. Case report: autochthonous case of human visceral leishmaniasis in the West Bank. Palestine Am J Trop Med Hyg. 2020;103:1490–2.

    Article  PubMed  Google Scholar 

  38. Lewis DJ. A taxonomic review of the genus Phlebotomus (Diptera: Psychodidae). Bull Br Mus Nat His. 1982;45:121–209.

    Google Scholar 

  39. Artemiev MM, Neronov VM. Distribution and Ecology of Sand flies of the Old World (Genus Phlebotomus). Moscow: Institute of Evolution, Morphology and Animal Ecology; 1984.

    Google Scholar 

  40. Lane R. Sand Flies (Phlebotomidae). London: RP Lane & RW Crosskey edn. Chapman- Hall; 1993.

    Google Scholar 

  41. Killick-Kendrick R, Tang Y, Killick-Kendrick M, Sang DK, Sirdar MK, Ke L, et al. The identification of female sand flies of the subgenus Larroussius by the morphology of the spermathecal ducts. Parassitologia. 1991;33:335–47.

    PubMed  Google Scholar 

  42. Casaril AE, de Oliveira LP, Alonso DP, de Oliveira EF, Gomes Barrios SP, de OliveiraMouraInfran J, et al. Standardization of DNA extraction from sand flies: application to genotyping by next generation sequencing. Exp Parasitol. 2017;177:66–72.

    Article  CAS  PubMed  Google Scholar 

  43. Corpet F. Multiple sequence alignment with hierarchical clustering. Nucleic Acids Res. 1988;16:10881–90.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Giantsis IA, Chaskopoulou A, Claude BM. Direct Multiplex PCR (dmPCR) for the Identification of six phlebotomine sand fly species (Diptera: Psychodidae), including major Leishmania vectors of the Mediterranean. J Econ Entomol. 2017;110:245–9.

    CAS  PubMed  Google Scholar 

  45. Early AM, Daniels RF, Farrell TM, Grimsby J, Volkman SK, Wirth DF, et al. Detection of low-density Plasmodium falciparum infections using amplicon deep sequencing. Malar J. 2019;18:219.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  46. Kumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol. 2018;35:1547–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. van Eys GJ, Schoone GJ, Kroon NC, Ebeling SB. Sequence analysis of small subunit ribosomal RNA genes and its use for detection and identification of Leishmania parasites. Mol Biochem Parasitol. 1992;51:133–42.

    Article  PubMed  Google Scholar 

  48. Meredith SE, Zijlstra EE, Schoone GJ, Kroon CC, van Eys GJ, Schaeffer KU, et al. Development and application of the polymerase chain reaction for the detection and identification of Leishmania parasites in clinical material. Arch Inst Pasteur Tunis. 1993;70:419–31.

    CAS  PubMed  Google Scholar 

  49. Rodgers MR, Popper SJ, Wirth DF. Amplification of kinetoplast DNA as a tool in the detection and diagnosis of Leishmania. Exp Parasitol. 1990;71:267–75.

    Article  CAS  PubMed  Google Scholar 

  50. Marfurt J, Niederwieser I, Makia ND, Beck HP, Felger I. Diagnostic genotyping of old and new World Leishmania species by PCR-RFLP. Diagn Microbiol Infect Dis. 2003;46:115–24.

    Article  CAS  PubMed  Google Scholar 

  51. Marfurt J, Nasereddin A, Niederwieser I, Jaffe CL, Beck HP, Felger I. Identification and differentiation of Leishmania species in clinical samples by PCR amplification of the miniexon sequence and subsequent restriction fragment length polymorphism analysis. J Clin Microbiol. 2003;41:3147–53.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. el Tai NO, Osman OF, el Fari M, Presber W, Schonian G. Genetic heterogeneity of ribosomal internal transcribed spacer in clinical samples of Leishmania donovani spotted on filter paper as revealed by single-strand conformation polymorphisms and sequencing. Trans R Soc Trop Med Hyg. 2000;94:575–9.

    Article  PubMed  Google Scholar 

  53. de Pita-Pereira D, Cardoso MA, Alves CR, Brazil RP, Britto C. Detection of natural infection in Lutzomyia cruzi and Lutzomyia forattinii (Diptera: Psychodidae: Phlebotominae) by Leishmania infantum chagasi in an endemic area of visceral leishmaniasis in Brazil using a PCR multiplex assay. Acta Trop. 2008;107:66–9.

    Article  PubMed  CAS  Google Scholar 

  54. de Pita-Pereira D, Alves CR, Souza MB, Brazil RP, Bertho AL, de Figueiredo BA, et al. Identification of naturally infected Lutzomyia intermedia and Lutzomyia migonei with Leishmania (Viannia) braziliensis in Rio de Janeiro (Brazil) revealed by a PCR multiplex non-isotopic hybridisation assay. Trans R Soc Trop Med Hyg. 2005;99:905–13.

    Article  PubMed  CAS  Google Scholar 

  55. Pereira LOR, Moreira RB, de Oliveira MP, Reis SO, de Oliveira Neto MP, Pirmez C. Is Leishmania (Viannia) braziliensis parasite load associated with disease pathogenesis? Int J Infect Dis. 2017;57:132–7.

    Article  PubMed  Google Scholar 

  56. Sales K, Miranda DEO, Paiva MHS, Figueredo LA, Otranto D, Dantas-Torres F. Fast multiplex real-time PCR assay for simultaneous detection of dog and human blood and Leishmania parasites in sand flies. Parasit Vectors. 2020;13:131.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Pareyn M, Hendrickx R, Girma N, Hendrickx S, Van Bockstal L, Van Houtte N, et al. Evaluation of a pan-Leishmania SL RNA qPCR assay for parasite detection in laboratory-reared and field-collected sand flies and reservoir hosts. Parasit Vectors. 2020;13:276.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Mota TF, Brodskyn CI, Morello LG, Marchini FK, Krieger MA, de Cassia Pontello Rampazzo R, et al. Multiplex qPCR assay to determine Leishmania infantum load in Lutzomyia longipalpis sandfly samples. Med Vet Entomol. 2022;36:176–84.

    Article  PubMed  Google Scholar 

  59. Degrave W, Fernandes O, Campbell D, Bozza M, Lopes U. Use of molecular probes and PCR for detection and typing of Leishmania-a mini-review. Mem Inst Oswaldo Cruz. 1994;89:463–9.

    Article  CAS  PubMed  Google Scholar 

  60. Eberhardt E, Van den Kerkhof M, Bulte D, Mabille D, Van Bockstal L, Monnerat S, et al. Evaluation of a pan-Leishmania spliced-leader RNA detection method in human blood and experimentally infected Syrian golden hamsters. J Mol Diagn. 2018;20:253–63.

    Article  CAS  PubMed  Google Scholar 

  61. Hitakarun A, Tan-ariya P, Siripattanapipong S, Mungthin M, Piyaraj P, Naaglor T, et al. Comparison of PCR methods for detection of Leishmania siamensis infection. Parasit Vectors. 2014;7:458.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  62. Patino LH, Castillo-Castaneda AC, Munoz M, Jaimes JE, Luna-Nino N, Hernandez C, et al. Development of an amplicon-based next-generation sequencing protocol to identify Leishmania species and other Trypanosomatids in leishmaniasis endemic areas. Microbiol Spectr. 2021;9:e0065221.

    Article  PubMed  Google Scholar 

  63. Castillo-Castaneda A, Patino LH, Munoz M, Ayala MS, Segura M, Bautista J, et al. Amplicon-based next-generation sequencing reveals the co-existence of multiple Leishmania species in patients with visceral leishmaniasis. Int J Infect Dis. 2022;115:35–8.

    Article  CAS  PubMed  Google Scholar 

  64. Chen S, Zhou Y, Chen Y, Huang T, Liao W, Xu Y, et al. Gencore: an efficient tool to generate consensus reads for error suppressing and duplicate removing of NGS data. BMC Bioinformatics. 2019;20:606.

    Article  PubMed  PubMed Central  Google Scholar 

  65. Erisoz Kasap O, Linton YM, Karakus M, Ozbel Y, Alten B. Revision of the species composition and distribution of Turkish sand flies using DNA barcodes. Parasit Vectors. 2019;12:410.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  66. Wijerathna T, Gunathilaka N. Morphological identification keys for adults of sand flies (Diptera: Psychodidae) in Sri Lanka. Parasit Vectors. 2020;13:450.

    Article  PubMed  PubMed Central  Google Scholar 

Download references


Not applicable.


The project was funded by the USAID (Grant M33-07).

Author information

Authors and Affiliations



SE and AN: conceptualization, wrote original draft; AJ: analyzed sequences and phylogenetics; MT and HJ: did the experimental work and prepared Figs. 1, 2, 3; AN, SE and IA: analyzed NGS work; SS: collected and identified sand flies; ZA: Project administration. All authors reviewed the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Suheir Ereqat.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Additional file 1: Table S1

. Reference DNA samples for Leishmania and sand fly species used in the study. Table S2. Sand flies and Leishmania GenBank accession numbers used in this study for primer designing. Table S3. The virtual probe sequences used for detection and identification of sand flies and Leishmania parasites.

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 The Creative Commons Public Domain Dedication waiver ( 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

Verify currency and authenticity via CrossMark

Cite this article

Nasereddin, A., Ereqat, S., Al-Jawabreh, A. et al. Concurrent molecular characterization of sand flies and Leishmania parasites by amplicon-based next-generation sequencing. Parasites Vectors 15, 262 (2022).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI:


  • Leishmania
  • Phlebotomine sand flies
  • Amp-NGS
  • Taxonomy