Abstract
BackgroundPost-kala-azar dermal leishmaniasis (PKDL) caused by Leishmania donovani (LD) is a skin disorder that often appears after treatment of visceral leishmaniasis (VL) patients. PKDL patients are potential reservoirs of LD parasites, which can initiate a new epidemic of anthroponotic VL. Therefore, host infectiousness to its sand fly vector is a critical factor for transmission, and its accurate estimation can facilitate control strategies. At present, conventional microscopy serves as the reference method to detect parasites in its vector. However, low sensitivity of microscopy can be a limiting factor.MethodsIn this study, real-time quantitative PCR (LD-qPCR) and recombinase polymerase amplification (LD-RPA) assays were evaluated against microscopy for the detection of LD DNA extracted from live sand flies five days after controlled feeding on PKDL cases.ResultsThe sensitivity of LD-qPCR and LD-RPA assays were found to be 96.43 and 100%, respectively, against microscopy for the selected fed sand flies (n = 28), and an absolute specificity of both molecular tools for apparently unfed sand flies (n = 30). While the proportion of infectious cases among 47 PKDL patients was estimated as 46.81% as defined by microscopic detection of LD in at least one fed sand fly per case, LD-RPA assay evaluation of only the microscopy negative sand flies fed to those 47 PKDL cases estimated an even greater proportion of infectious cases (51.06%). In overall estimation of the infectious cases in retrospective manner, discordance in positivity rate was observed (p < 0.05) between LD-RPA (59.57%) assay and microscopy (46.81%), while LD-RPA had slightly better positivity rate than LD-qPCR (55.32%) as well.ConclusionsConsidering the sensitivity, cost, detection time, and field applicability, RPA assay can be considered as a promising single molecular detection tool for investigations pertaining to LD infections in sand flies and/or host infectiousness in PKDL, while it can also be useful in confirmation of microscopy negative sand fly samples.Graphical abstract
Highlights
Post-kala-azar dermal leishmaniasis (PKDL) caused by Leishmania donovani (LD) is a skin disorder that often appears after treatment of visceral leishmaniasis (VL) patients
We have shown in our previous XD study with quantitative polymerase chain reaction of skin punch biopsy that parasite load is associated with infectiousness of PKDL patients to sand fly and can be a promising surrogate or complementary marker of onward transmission potential [10]
We evaluated the performance of LDqPCR and LD-recombinase polymerase amplification (RPA) assays against microscopy for detection of LD in DNA samples extracted from sand flies that were fed on PKDL patients
Summary
Post-kala-azar dermal leishmaniasis (PKDL) caused by Leishmania donovani (LD) is a skin disorder that often appears after treatment of visceral leishmaniasis (VL) patients. PKDL patients are prone to parasite uptake if bitten by its sand fly vector, because typical PKDL manifestation is in the form of painless macular or papulo-nodular lesions, or a mix of both, that harbour parasites This may play a major role in the transmission cycle of leishmaniasis [5]. The advantage of XD lies in its capability of utilizing the insect vector as biological culture medium to amplify an inoculum of living parasites even with very low parasite load, constituting irrefutable evidence of viable and transmissible LD infection This information can be especially important to evaluate host and/or vector infectiousness status in epidemiological surveillance and to assess the effect of chemotherapeutics on the reservoir potential and their efficacy in treated cases of PKDL patients without invasive procedures to establish whether the infection has healed or not [8]
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