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Development and evaluation of a loop-mediated isothermal amplification assay for rapid detection of Leishmania amazonensis in skin samples

Celeste, Jordanna Luiza de Lima, Caldeira, Roberta Lima, Pires, Simone da Fonseca, Silveira, Karine Dias, Soares, Rodrigo Pedro, de Andrade, Hélida Monteiro
Experimental parasitology 2019 v.203 pp. 23-29
Leishmania amazonensis, diagnostic techniques, dogs, etiological agents, gene amplification, humans, kinetoplast DNA, loop-mediated isothermal amplification, parasite load, parasites, quantitative polymerase chain reaction, rapid methods, visceral leishmaniasis, Brazil
In Brazil, Leishmania amazonensis is one of the etiological agents of tegumentary leishmaniasis and can cause a wide spectrum of diseases in humans, resulting in cutaneous, mucosal, diffuse, and even visceral leishmaniasis. Besides, this species has also been reported to affect dogs, causing typical symptoms of visceral disease. Unfortunately, the diagnostic of the Leishmania species is not routinely performed due to the difficulties of the available methods. In view of this, different molecular methods have been used in an attempt to solve the problem of diagnosis. Loop-mediated isothermal amplification (LAMP) is a relatively new nucleic acid amplification method, which has been successfully applied in the diagnosis of Leishmania spp. infections. However, this is the first work that standardizes a specific LAMP reaction for L. amazonensis. The set of primers selected were designed from the kDNA minicircle sequence of the L. amazonensis (GenBank: U19810.1). The LAMP assay developed in the present study showed 100% specificity and 89% sensitivity when compared with conventional PCR and was more sensitive than qPCR. In addition, the LAMP reaction developed here was able to amplify a qPCR sample with a parasite load of only 28 parasites in 50 ng of DNA. Consequently, considering the LAMP reaction specific to L. amazonensis and several advantages of the method (such as high efficiency, sensitivity and specificity), we believe that this reaction can be used as a promising diagnostic tool in clinical practice, field studies, and research.