A loop-mediated isothermal amplification (LAMP) kit for molecular detection of Trypanosoma cruzi DNA; a feasibility study

SUSANA A. BESUSCHION; ALBERT PICADO; ALEJANDRO BENATAR; JOSEPH D'UNGU; ALEJANDRO SCHIJMAN; MÓNICA LLANO MURCIA ; CONCEPCION PUERTA

Barcelona

Taller; Taller Importado de Enfermedad de Chagas; 2016

Instituto de Salud Global Barcelona

Loop-mediated isothermal amplification (LAMP) tests are currently used as point-of care (POC) molecular tests in neglected parasitic diseases, such as leishmaniasis or sleeping sickness. A LAMP test for Trypanosoma cruzi, the etiological agent of Chagas Disease (ChD), would allow a rapid and reliable diagnosis, in particular in cases of acute and congenital ChD (CChD).We evaluated the performance a Trypanosoma cruzi LAMP kit using purified DNA, spiked blood and clinical specimens. Quantitative PCR (qPCR) was used as a reference. Different extraction methods for LAMP were also evaluated. The LAMP reaction was performed at 62.5°C for 45 min. Analytical sensitivity was measured in ten-fold dilutions of CL Brener (TcVI) and Silvio X10 (TcI) DNA. Analytical specificity was measured using ten-fold dilutions of different Leishmania species and Trypanosoma rangeli DNAs as well as non-infected human DNA. Seronegative blood in EDTA (EB) or heparine (HB) was spiked with ten-fold dilutions of CL Brener. EB spiked blood was also used as dried blood spot (DBS). Stored DNA from EB clinical samples was tested: 4 Congenital ChD cases, 5 Chronic ChD cases with low parasitic loads, 10 immunosuppressed ChD patients and 5 seronegative controls. DNA extraction was done with a commercial kit (EB, HB and DBS samples) and using the boil & spin (B&S) method (HB samples only).The T. cruzi LAMP kit showed better analytical sensitivity than qPCR in purified DNA specimens, especially for TcI DNA. Analytical sensitivity was 10-2 and 10 -1 par.eq/mL from spiked EB and HB extracted by columns, respectively, and 10-2 par.eq/mL from HB using B&S. The analytical sensitivity in DBS samples was 10-2 par.eq/mL. The T. cruzi LAMP was positive in congenital and immunosuppressed ChD samples spanning from 4.8 to 3684 par.eq/ml, in agreement with qPCR. Chronic ChD samples were only detectable by qPCR, with Ct values below the limit of quantification. The kit was specific for T.cruzi DNA and samples from seropositive patients.Preliminary results encourage the potential use of T. cruzi LAMP as a POC test for CChD and Chagas reactivation.