Evaluation of molecular assays to detect Leishmania donovani in Phlebotomus argentipes fed on post-kala-azar dermal leishmaniasis patients

Abstract

Background 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 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. Methods In 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. Results The 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. Conclusions Considering 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

Detection of Parasites in the Field: The Ever-Innovating CRISPR/Cas12a

“…Isothermal amplification has been employed in the diagnosis of various parasitic diseases, addressing the challenges posed by traditional diagnostic methods [ 42 , 43 , 44 , 45 , 46 ]. Compared to PCR, isothermal amplification technology, exemplified by loop-mediated isothermal amplification (LAMP) and RPA, significantly reduces the reaction time and dependence on instruments.…”

“…Compared to PCR, isothermal amplification technology, exemplified by loop-mediated isothermal amplification (LAMP) and RPA, significantly reduces the reaction time and dependence on instruments. RPA is an efficient method for on-site detection due to its simple primer design, low-temperature requirements, and easy storage [ 43 , 44 ]. Recombinase-aid amplification (RAA), based on the same principle, also offers these advantages in rapid detection [ 47 ].…”

“…Although these techniques have been instrumental in establishing dependable diagnostic methods for parasitosis, including malaria, filariasis, toxoplasmosis, and echinococcosis, they require prolonged reaction times, intricate handling, expensive laboratory equipment, and a high level of technical expertise [10,[38][39][40][41]. Isothermal amplification has been employed in the diagnosis of various parasitic diseases, addressing the challenges posed by traditional diagnostic methods [42][43][44][45][46]. Compared to PCR, isothermal amplification technology, exemplified by loop-mediated isothermal amplification (LAMP) and RPA, significantly reduces the reaction time and dependence on instruments.…”

Effects of Synthetic Ligustrazine-Based Chalcone Derivatives on Trypanosoma brucei brucei and Leishmania spp. Promastigotes

“…The presence of the sand fly Phlebotomus papatasi in regions where L. major infections are common is likely to blame for the vast majority of reported cases of the disease in Saudi Arabia and other Arabian countries [ 13 ]. The zoonotic form of CL evolved as a result of the global expansion of leishmaniasis in the 20th century, with an estimated yearly incidence in Saudi Arabia of more than 4000 [ 14 , 15 ]. The widespread prevalence of desert rodents (reservoir animals) and sand flies is largely responsible for the endemic nature of CL in several regions in Saudi Arabia.…”

Handheld Ultra-Fast Duplex Polymerase Chain Reaction Assays and Lateral Flow Detection and Identification of Leishmania Parasites for Cutaneous Leishmaniases Diagnosis

“…However, this test was not recommended for use by some studies [17]; or it was shown that it should be complemented by additional methods because of its low sensitivity [18]. Other POC format tools based on isothermal amplification were also developed [19][20][21][22][23][24]. Nevertheless, all these tools are generic and detect Leishmania parasites without identifying them.…”