A loop-mediated is othermal amplification (LAMP) kit for molecular detection of Trypanosoma cruzi dna: A feasibility study

Abstract

Loop-mediated isothermal amplification (LAMP) tests have been developedas molecular tests for neglected parasitic diseases such as leishmaniasisor sleeping sickness. A LAMP test for Trypanosoma cruzi, the etiologicalagent of Chagas Disease (ChD), would allow a rapid and reliable diagnosis,in particular in cases of acute and congenital ChD (CChD). We evaluatedthe performance a Trypanosoma cruzi LAMP kit using purified DNA, spikedblood and clinical specimens. Quantitative PCR (qPCR) was used as areference. Different extraction methods for LAMP were also evaluated. TheLAMP reaction was performed at 62.5°C for 45 min. Analytical sensitivitywas measured in ten-fold dilutions of CL Brener (TcVI) and Silvio X10(TcI) DNA. Analytical specificity was measured using ten-fold dilutions ofdifferent Leishmania species and Trypanosoma rangeli DNAs as well asnon-infected human DNA. Seronegative blood in EDTA (EB) or heparin (HB)was spiked with ten-fold dilutions of CL Brener. EB spiked blood was alsoused as dried blood spot (DBS). Stored DNA from EB clinical samples wastested, including 4 Congenital ChD cases, 5 Chronic ChD cases with lowparasite loads, 10 immunosuppressed ChD patients and 5 seronegativecontrols. DNA extraction was done with a commercial kit (EB, HB and DBSsamples) and using the boil & spin (B&S) method (HB samples only). TheT. cruzi LAMP kit showed better analytical sensitivity than qPCR in purifiedDNA specimens, especially for TcI DNA. Analytical sensitivity was 10-2 and10-1 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 DBSsamples was 10-2 par.eq/mL. T. cruzi LAMP was positive in congenital andimmunosuppressed ChD samples spanning from 4.8 to 3,684 par.eq/ml,in agreement with qPCR. Chronic ChD samples were only detectable byqPCR, with Ct values below the limit of quantification. The kit was specificfor T. cruzi DNA and samples from seropositive patients.