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

Abedelmajeed Nasereddin, Suheir Ereqat*, Amer Al-Jawabreh, Mohamad Taradeh, Ibrahim Abbasi, Hanan Al-Jawabreh, Samer Sawalha, Ziad Abdeen

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

2 Scopus citations


Background: 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. Methods: 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. Results: 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%). Conclusions: 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: [Figure not available: see fulltext.]

Original languageAmerican English
Article number262
Pages (from-to)1-12
JournalParasites and Vectors
Issue number1
StatePublished - Dec 2022
Externally publishedYes

Bibliographical note

Publisher Copyright:
© 2022, The Author(s).


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


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