Evaluation of Loop-mediated isothermal amplification for detection of Trypanosoma cruzi in clinical samples.

BISIO MMC; LACRUZ A; GUEVARA P; SCHIJMAN AG

Mar del Plata

Congreso; IX Congreso de Protozoología y Enfermedades Parasitarias; 2011

Sociedad Argentina de Protozoología

Anti-parasitic treatment is indicated in congenital Chagas infection (CCI) with greater success in newborns closer to delivery. However, the lack of access to reliable point-of-care diagnostic tests contributes enormously to the under-diagnosis of CCI in newborns without symptoms of disease. Conventional diagnosis of CCI relies on microscopic observation of bloodstream trypomastigotes such as the microhaematocrit (MH) assay. However, it is time-consuming, highly operator-dependent leading to variability in sensitivity. PCR has appeared as a promising sensitive tool but it is not applicable to field laboratories due the requirement of expensive equipment, specialized infrastructure and skilled personnel. Loop-mediated isothermal amplification (LAMP) is an established nucleic acid amplification method promising rapid, accurate, and cost-effective diagnosis of infectious diseases in a simple one tube format. LAMP requires only a water bath and it can be conducted within 60 minutes. We optimized a LAMP assay based on T. cruzi 18S ribosomal RNA genes using previously reported primer sequences1 to allow its use in the field. For this purpose, hidroxinaftol blue (HNB) was added to the reaction tube2 to enable direct detection by the naked eye avoiding the need of  equipment for visualization of the amplicons. LAMP analytical specificity was tested with DNA from T. rangeli and L. brasiliensis. The ability to detect strains from the 6 discrete typing units (DTUs) was also demonstrated. The detection limit was 80 fg of T. cruzi Cl Brener DNA per reaction-tube. The standardized reaction was then validated with DNA from clinical samples characterised by MH or serology, kDNA-PCR and, in some cases Real Time PCR (qPCR). Seven blood samples from newborns and 10 from chronically infected patients were analyzed. Indeed, samples from all 3 CCI cases (qPCR>50par/ml) allowed positive LAMP results whereas all 4 tested non-infected babies were LAMP negative. Out of the chronically infected patients, only 1/5 paediatric patients was LAMP positive (qPCR 8.3 par/ml) whereas 5/5 pregnant women yielded negative results. Our work brings this LAMP strategy as a potential test for diagnosis of CCI in field work or resource-limited laboratories. Future research including the design of primer sequences targeted to highly repetitive genes and its validation in field work are needed to establish the potential of this technology for diagnosis of Chagas disease.