Development of a novel ddPCR assay for the simultaneous detection of the protozoan parasites Leishmania infantum and Leishmania tarentolae

Abstract

Background: Leishmaniases, caused by protozoan parasites of the genus Leishmania, are vector-borne diseases occurring mainly in the tropics and subtropics of the world, as well as in the Mediterranean Basin. In this area, the mammalian pathogen Leishmania infantum is endemic, along with the reptile-associated Leishmania tarentolae. The two species occur in sympatry, and there is evidence that the exposure to L. tarentolae in mammalian hosts may elicit a protective immune response towards pathogenic Leishmania species. Accurate detection methods for both species are therefore crucial for gathering comprehensive information on the epidemiology of leishmaniases. In microbiological diagnosis, limits in detection performance imply the risk of false negatives and other issues, which highlights the need for sensitive methods.

Methods: Here, we developed a droplet digital polymerase chain reaction assay targeting the kinetoplast minicircle DNA, for the simultaneous and differential detection of L. infantum and L. tarentolae. The assay features primers designed to bind to both species and species-specific probes. The assay was validated on three cultured isolates for each species, whose cells were spiked into Leishmania-negative dog blood, and on Leishmania-positive sand flies. Sensitivity was assessed with testing serial dilutions, and specificity was evaluated by assessing the cross-reactivity of the probes with the controls of Leishmania-free dog blood and male sand fly DNA.

Results: The assay demonstrated high sensitivity, with a limit of detection corresponding to one Leishmania cell in the reaction mix for isolates of both L. infantum and L. tarentolae. Limited cross-reaction of the L. tarentolae-targeting probe was observed on L. infantum isolates. No cross-reaction was observed with the controls of Leishmania-free dog blood and male sand flies.

Conclusions: The protocol can represent a valuable method for comprehensive surveillance in both canine hosts and sand flies in areas in which L. infantum and L. tarentolae occur in sympatry.

Keywords: Leishmania infantum; Leishmania tarentolae; Droplet digital PCR; Epidemiology; Leishmaniasis; Sand flies.

Fig. 1

ddPCR assay sensitivity assessment on dog blood spiked with serial dilutions of the commercial P10 L. tarentolae strain (Jena Bioscience), and the wild “W” and “RI325” L. tarentolae strains. Annealing temperature of 55 °C. The limit of detection is 0.2 Leishmania cells/μl of DNA extracted from dog blood added to the ddPCR mix, corresponding to one Leishmania cell present in the ddPCR mix. The fluorescence amplitude intensity is indicated on the y-axis. Each dot represents an individual droplet. The pink line represents the amplitude threshold above which a droplet is assigned as positive (i.e. it contains one or more target copies, coloured in blue), and below which a droplet is assigned as negative (i.e. it contains no target copies, coloured in grey). For each sample, the number of Leishmania cells per μL of input DNA sample is shown above, while the concentration of target DNA copies per μL of input DNA sample is displayed in the box below

Fig. 2

ddPCR assay sensitivity assessment on dog blood spiked with serial dilutions of the L. infantum “MHOM/TN/80/IPT-1” reference strain (OIE Reference Laboratory National Reference Center for Leishmaniasis—C.Re.Na.L., Palermo, Italy), and the wild “S” and “G” L. infantum strains (University of Bari Aldo Moro). Annealing temperature of 50 °C. The limit of detection is 0.2 Leishmania cells/μl of DNA extracted from dog blood added to the ddPCR mix, corresponding to one Leishmania cell present in the ddPCR mix. The fluorescence amplitude intensity is indicated on the y-axis. Each dot represents an individual droplet. The pink line represents the amplitude threshold above which a droplet is assigned as positive (i.e. it contains one or more target copies, coloured in green), and below which a droplet is assigned as negative (i.e. it contains no target copies, coloured in grey). For each sample, the number of Leishmania cells per μL of input DNA sample is shown above, while the concentration of target DNA copies per μL of input DNA sample is displayed in the box below

Fig. 3

ddPCR specificity assessment by testing for cross-reaction between the FAM probe and the L. infantum DNA extracted from the three isolates used in the study. Annealing temperature of 50 °C. A maximum of four FAM-positive droplets were detected when analysing L. infantum samples. Therefore, any result with fewer than five FAM-positive droplets should be considered negative. The fluorescence amplitude intensity is indicated on the y-axis. Each dot represents an individual droplet. The pink line represents the amplitude threshold above which a droplet is assigned as positive (i.e. it contains one or more target copies, coloured in blue), and below which a droplet is assigned as negative (i.e. it contains no target copies, coloured in grey)

Fig. 4

ddPCR assay specificity assessment on the controls represented by DNA extracted from Leishmania-negative dog blood and male sand flies. Annealing temperature of 50 °C. Each sample has been tested with both the FAM probe, targeting L. tarentolae, and with the HEX probe, targeting L. infantum. A maximum of two FAM-positive droplets have been observed in the sand fly samples, whereas no droplets hybridized with the HEX probe. Given the threshold set after testing for cross-reaction between the FAM probe and the L. infantum DNA, the control samples should be considered negative. For each sample, the FAM result is shown in the box on the top, while the HEX result is displayed in the box below. The fluorescence amplitude intensity is indicated on the y-axis. Each dot represents an individual droplet. The pink line represents the amplitude threshold above which a droplet is assigned as positive (i.e. it contains one or more target copies, coloured in blue), and below which a droplet is assigned as negative (i.e. it contains no target copies, coloured in grey)

Fig. 5

Results of the ddPCR testing of the qPCR-Leishmania-positive sand flies from the collection of the University of Bari Aldo Moro. Annealing temperature of 50 °C. Each sample has been tested with both the FAM probe, targeting L. tarentolae, and with the HEX probe, targeting L. infantum. For each sample, the FAM result is shown in the box on the top, while the HEX result is displayed in the box below. Sample names are indicated at the bottom of the HEX box. The fluorescence amplitude intensity is indicated on the y-axis. Each dot represents an individual droplet. The pink line represents the amplitude threshold above which a droplet is assigned as positive (i.e. it contains one or more target copies, coloured in blue or green), and below which a droplet is assigned as negative (i.e. it contains no target copies, coloured in grey)