EVALUATION OF A KIT FOR MOLECULAR DETECTION OF TRYPANOSOMA CRUZI DNA BASED ON LOOP MEDIATED ISOTHERMAL AMPLIFICATION (LAMP)

SUSANA ALICIA BESUSCHIO; ALEJANDRO BENATAR ; CONCEPCIÓN PUERTA ; MÓNICA LLANO MURCIA ; ISRAEL CRUZ MATA ; ALEJANDRO G. SCHIJMAN ; ALBERTO PICADO DE PUIG; MARÍA DE LOS ANGELES CURTO; JOSEPH NDUNGU

Santa Fe

Congreso; XXVIII Reunión Anual de la Sociedad Argentina de Protozoologia y Enfermedades Parasitarias; 2016

SAP

Introduction: Loop-mediated isothermal amplification (LAMP) is a molecular technology platform developed at Eiken Chemical Company (http://www.eiken.co.jp/en)designed for point-of-care diagnosis. Aims: This work aimed to evaluate sensitivity and specificity of Loopamp? Trypanosoma cruzi kit using purified DNA, spiked blood and clinical specimens compared to quantitative PCR. Methods: LAMP reaction designed with dried reagents inside the cap of the tube, with primers targeted to T.cruzi satellite repeats, was performed at 62.5°C for 45 min and visualized by fluorescence, naked eye and agarose gel electrophoresis. Analytical sensitivity was measured in ten-fold dilutions of CLBrener (TcVI) and Silvio X10 (TcI) DNA (10 exp 3-10 exp¯3 fg/ul) and compared to TaqMan duplex qPCR. Analytical specificity was measured using ten-fold dilutions of Leishmania mexicana, L. donovani, L.major, L. chagasi and Trypanosoma rangeli DNAs (10 exp 4 -10 fg/ul) and non-infected human DNA. Spiked blood analysis: Seronegative blood was collected in EDTA (EB) or heparine (HB) and spiked with ten-fold dilutions of CL Brener (10 exp 3 -10 exp -3 parasite equivalents/mL).EB-DNA was extracted using a commercial kit (Roche Diagnostics) and HB-DNA using that kit or boil&spin procedure. Stored DNA from EB clinical samples was tested: Congenital Chagas disease newborns (CCD N= 24 ), immunosuppressed CD patients due to organ transplantation (N= 31), AIDS (N= 4 in cerebrospinal fluid and EB) and seronegative controls (N=37). Results: LAMP detected up to 0.01 fg/ul of TcVI and TcI DNA, whereas qPCR detected 0.1 fg/ul of TcVI DNA and 1 fg/ul of TcI DNA triplicates. Analytical sensitivity was 10 exp-2 and 10 exp -1 par.eq/mL from spiked EB and HB extracted by columns, respectively, and 10 exp -2 par.eq/mL from HB using boil&spin LAMP detected CCD and immunosuppressed CD EB samples spanning 4.82.5 to 3684 par.eq/ml, in agreement with qPCR. The kit was specific for T.cruzi DNA and samples from seropositive patients. Conclusions: The Loopamp? Trypanosoma cruzi kit showed better analytical sensitivity than qPCR in purified DNA specimens, especially for TcI DNA, whereas in clinical samples optimization of DNA extraction for LAMP must be improved yet. Work is currently undergone in this direction. Preliminary results encourage its potential application in early diagnosis of CCD and Chagas reactivation.