title: Development and evaluation of a loop-mediated isothermal amplification assay for rapid detection of Leishmania infantum in canine leishmaniasis based on cysteine protease B genes.
abstract: We developed a Leishmania infantum specific LAMP assay that was carried out using a set of, six primers targeting the cysteine protease B multi copy gene of L. infantum. Our result shows that we, successfully detect the L. infantum DNA and that amplification is specific as no cross reaction was seen, with L. major, L. tropica, L. turanica, L. aethiopica, L. tarentolae, L. gerbilii, Trypanosoma cruzi or, human genomic DNA. When compared to conventional cpb based PCR, the sensitivity of LAMP assay, was higher with a detection limit of 50 fg/μl of genomic L. infantum parasite DNA. Accurate and rapid, diagnosis of canine leishmaniasis (CanL) is an important issue that allows early treatment and, prevents transmission. Our developed LAMP assay was used to evaluate occurrences of Leishmania infantum in seventy five (75) dogs from the field. Blood samples were used to perform LAMP assay, classical PCR, IFAT and microscopy that was used as gold standard. The IFAT in addition to, microscopy, are the basic techniques used for CanL diagnosis at the School of Veterinary Medicine, where we obtained our samples. Compared to molecular methods, the serology (IFAT) test shows the, best sensitivity (88.57%) with, however, a much lower specificity (52.5%) due to a relatively high, number of false-positive results (22 animals). The PCR assay shows a low sensitivity (37.14%) and, specificity around (82.5%). Our LAMP assay shows a suitable sensitivity (54%) and a good specificity, (80%), with however, positive (70%) and negative (66%) predictive values. Furthermore, the best, positive likelihood ratio (LR+) was obtained by LAMP assay (2.7). This technique presents the highest, kappa value (with a fair agreement of 0.34). Moreover, the relative stability of the reagents indicates, that LAMP may be a good alternative to a conventional PCR, especially under field conditions. Finally in, a brief cost evaluation, the LAMP assay compares favorably with other molecular diagnostic tests. This, is the first study that evaluates the L. infantum specific LAMP alongside other diagnostics tools for, CanL. Our results indicate a suitable sensitivity and specificity for the developed LAMP assay that could, has usefulness application on dogs and human L. infantum diagnosis.
authors: Institut Pasteur de Tunis, Laboratoire d'Epidémiologie et d'Ecologie Parasitaire (LR00SP04), Tunis, Tunisia.
authors: Melek
authors: M
authors: Chaouch
authors: Moez
authors: M
authors: Mhadhbi
authors: Emily R
authors: ER
authors: Adams
authors: Gerard J
authors: GJ
authors: Schoone
authors: Sassi
authors: S
authors: Limam
authors: Zyneb
authors: Z
authors: Gharbi
authors: Mohamed Aziz
authors: MA
authors: Darghouth
authors: Ikram
authors: I
authors: Guizani
authors: Souha
authors: S
authors: BenAbderrazak
mesh_terms: D000818:Animals; D057056:Cysteine Proteases / Q000235:genetics / Q000378:metabolism*; D004283:Dog Diseases / Q000175:diagnosis / Q000469:parasitology*; D004285:Dogs; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D018314:Leishmania infantum / Q000302:isolation & purification*; D007898:Leishmaniasis, Visceral / Q000175:diagnosis / Q000469:parasitology / Q000662:veterinary*; D008297:Male; D008853:Microscopy; D021141:Nucleic Acid Amplification Techniques / Q000662:veterinary*; D016133:Polymerase Chain Reaction / Q000662:veterinary; D012680:Sensitivity and Specificity; D013997:Time Factors
chemical_list: D057056:Cysteine Proteases
keywords: Diagnosis; Dog; LAMP; Leishmania infantum; Tunisia; cpb