Analytical sensitivity and specificity of a loop-mediated isothermal amplification (LAMP) kit prototype for detection of Trypanosoma cruzi DNA in human blood samples

Abstract

This study aimed to assess analytical parameters of a prototype LAMP kit that was designed for detection of Trypanosoma cruzi DNA in human blood. The prototype is based on the amplification of the highly repetitive satellite sequence of T. cruzi in microtubes containing dried reagents on the inside of the caps. The reaction is carried out at 65°C during 40 minutes. Calcein allows direct detection of amplified products with the naked eye. Inclusivity and selectivity were tested in purified DNA from Trypanosoma cruzi stocks belonging to the six discrete typing units (DTUs), in DNA from other protozoan parasites and in human DNA. Analytical sensitivity was estimated in serial dilutions of DNA samples from Sylvio X10 (Tc I) and CL Brener (Tc VI) stocks, as well as from EDTA-treated or heparinized blood samples spiked with known amounts of cultured epimastigotes (CL Brener). LAMP sensitivity was compared after DNA extraction using commercial fiberglass columns or after “Boil & Spin” rapid preparation. Moreover, the same DNA and EDTA-blood spiked samples were subjected to standardized qPCR based on the satellite DNA sequence for comparative purposes. A panel of peripheral blood specimens belonging to Chagas disease patients, including acute, congenital, chronic and reactivated cases (N = 23), as well as seronegative controls (N = 10) were evaluated by LAMP in comparison to qPCR. LAMP was able to amplify DNAs from T. cruzi stocks representative of the six DTUs, whereas it did not amplify DNAs from Leishmania sp, T. brucei sp, T. rangeli KPN+ and KPN-, P. falciparum and non-infected human DNA. Analytical sensitivity was 1x10 ^-2 fg/μL of both CL Brener and Sylvio X10 DNAs, whereas qPCR detected up to 1x 10 ⁻¹ fg/μL of CL Brener DNA and 1 fg/μl of Sylvio X10 DNA. LAMP detected 1x10 ^-2 parasite equivalents/mL in spiked EDTA blood and 1x10 ^-1 par.eq/mL in spiked heparinized blood using fiberglass columns for DNA extraction, whereas qPCR detected 1x10 ^-2 par.eq./mL in EDTA blood. Boil & Spin extraction allowed detection of 1x10 ^-2 par.eq /mL in spiked EDTA blood and 1 par.eq/ml in heparinized blood. LAMP was able to detect T. cruzi infection in peripheral blood samples collected from well-characterised seropositive patients, including acute, congenital, chronic and reactivated Chagas disease. To our knowledge, this is the first report of a prototype LAMP kit with appropriate analytical sensitivity for diagnosis of Chagas disease patients, and potentially useful for monitoring treatment response.

Author summary

Trypanosoma cruzi, a parasite transmitted to humans from hematophagous insects, causes Chagas Disease, a Neglected Tropical Disease with public health impact, affecting 7 million people in Latin America. Although mainly related to low income populations inhabiting rural environments, migrations have conveyed Chagas Disease to urban areas of endemic and non-endemic countries. It often presents non-specific symptoms, and direct, low cost microscopy-based diagnosis only detects acute infections, missing a high proportion of cases. Serology is the “gold standard” diagnostic technique for chronic stages and needs the concordance of at least two different assays to confirm infection. In this context, we aimed to evaluate the analytical sensitivity and specificity of a prototype kit based on a novel and rapid molecular biology reaction, named Loop mediated isothermal amplification (LAMP), using standardized Real Time PCR as a comparator. To our knowledge, this is the first LAMP prototype kit with an analytical performance appropriate for human diagnosis of Chagas disease and potentially useful for monitoring treatment response. Its simple handling using basic laboratory devices will enable point-of-care diagnosis and screening for congenital infection at birth as well as early detection of acute infections due to oral contamination.