Leishmaniasis remains a significant public health concern. Accurate and efficient diagnostic methods are crucial for timely intervention and management. The limitations of current diagnostic approaches necessitated the exploration of novel targets and tools. This study describes 2 tandemly repeated sequences within the Leishmania genome that allow for sensitive and specific pan-Leishmania detection and speciation via quantitative polymerase chain reaction (qPCR) and recombinase polymerase amplification (RPA) and sequencing.MethodsThrough bioinformatic analysis of highly repeated elements of all Leishmania sp genomes, pan-Leishmania qPCR targets (CL3 and CL179) were identified and validated by qPCR. Amplicons from an RPA-based assay for CL179 qPCR with DNA from clinical samples were sequenced by nanopore sequencing.ResultsThe limits of detection for CL3 and CL179 qPCRs were ∼96 and ∼217 fg of genomic DNA across available Leishmania spp known to infect humans. The CL3 and CL179 qPCR could detect a single L donovani–infected macrophage. When tested with DNA extracted from skin biopsies (n = 8) and swabs (n = 7) from patients confirmed positive by 18S qPCR, CL3 and CL179 exhibited 100% sensitivity. Microbiopsies (n = 9) were also positive, with sensitivities of 88% and 67% for CL3 and CL179 qPCRs, respectively. Nanopore sequencing of RPA products resulted in the accurate speciation of Leishmania strains in patients confirmed to have leishmaniasis.ConclusionsCL3 and CL179 demonstrated high sensitivity and specificity, showing no cross-reactivity with related parasitic intracellular protists. Our data provide an ultrasensitive approach to the rapid diagnosis and subsequent speciation of all Leishmania strains known to infect humans.

Leishmaniasis remains a significant public health concern. Accurate and efficient diagnostic methods are crucial for timely intervention and management. The limitations of current diagnostic approaches necessitated the exploration of novel targets and tools. This study describes two tandemly repeated sequences within the Leishmania genome that allow for sensitive and specific Pan-Leishmania detection and speciation via qPCR and Recombinase Polymerase Amplification (RPA) and sequencing.MethodsThrough bioinformatic analysis of highly repeated elements of all Leishmania spp. genomes, pan-Leishmania qPCR targets (CL3 and CL179) were identified and validated by qPCR. Amplicons from an RPA-based assay for CL179 qPCR with DNA from clinical samples were sequenced by Nanopore sequencing.ResultsThe limits of detection for CL3 and CL179 qPCRs were ∼96 fg and ∼217 fg of gDNA - across available Leishmania spp. known to infect humans. The CL3 and CL179 qPCR could detect a single L. donovani-infected macrophage. When tested with DNA extracted from skin biopsies (n=8) and swabs (n=7) from patients confirmed positive by 18S qPCR, both CL3 and CL179 exhibited 100% sensitivity. Microbiopsies (n=9) were also positive, with sensitivities of 88% and 67% for CL3 and CL179 qPCRs respectively. Nanopore™ sequencing of RPA products resulted in the accurate speciation of Leishmania strains in patients confirmed to have leishmaniasis.ConclusionsBoth CL3 and CL179 demonstrated high sensitivity and specificity, showing no cross-reactivity with other related parasitic intracellular protists. Our data provides an ultrasensitive approach to the rapid diagnosis and subsequent speciation of all Leishmania strains known to infect humans.