Development of a Colorimetric Loop-Mediated Isothermal Amplification Assay for the Detection of Trypanosoma cruzi in Low-Resource Settings

Abstract

Chagas disease is an inflammatory parasitic infection caused by Trypanosoma cruzi (T. cruzi). Early diagnosis is crucial in guiding treatment and slowing disease progression; however, current diagnostic methods have insufficient detection limits and often require skilled technicians. Molecular tests, especially isothermal nucleic acid assays, are advantageous due to their excellent sensitivity, specificity, speed, and simplicity. Here, we optimized a colorimetric loop-mediated isothermal amplification (LAMP) assay for T. cruzi. We can detect as few as 2 genomic copies/reaction using three different T. cruzi strains. We examined selectivity using other parasitic protozoans and successfully detected T. cruzi DNA extracted from parasites in human whole blood down to 1.2 parasite equivalents/reaction. We also performed a blinded study using canine blood samples and established a 100% sensitivity, specificity, and accuracy for the colorimetric LAMP assay. Finally, we used a heated 3D printer bed and an insulated thermos cup to demonstrate that the LAMP incubation step could be performed with accessible, low-cost materials. Altogether, we have developed a high-performing assay for T. cruzi with a simple colorimetric output that would be ideal for rapid, low-cost screening at the point of use.

Keywords: Chagas disease; Trypanosoma cruzi; colorimetric output; loop-mediated isothermal amplification.

Figure 1

T. cruzi LAMP assay LOD and selectivity. Representative end-point colorimetric results (top), graphic demonstrating relationship between color (right) and hue value (y-axis), and quantified hue values (center) are shown for each data set. On a color wheel, a yellow hue (positive) is assigned 45–90° while pink (negative) is 315–360°. (A) Hue values for strain Dm28c are statistically significant down to 2 copies/reaction when compared to NTC (0). (B) Measured hue values for strain G show a statistically significant difference down to 2 copies/reaction when compared to NTC. (C) Hue values for strain CL are statistically significant down to 2 copies/reaction when compared to NTC. (D) Measured hue values for C. parvum, L. infantum, P. falciparum, and NTC show a statistically significant difference when compared to T. cruzi. n = 8 each circle represents a replicate; **** indicates p ≤ 0.0001.

Figure 2

Detection of T. cruzi DNA extracted from parasites in human whole blood. Highlighted for each data set are quantified hue values (center), graphic demonstrating relationship between color (right) and hue value (y-axis), and end-point colorimetric results (bottom). Each column in the 96-well plate is aligned with the corresponding concentration on the x-axis of the graph above. (A) DNA extraction was performed using the modified NucliSENS easyMAG Kit. There is a statistically significant difference in the hue value of LAMP products from as few as 1.2 parasite equivalents/reaction as compared to the negative control (0). (B) DNA extraction was conducted with the MagMAX Kit. There is a statistically significant difference in hue value down to 1.2 parasite equivalents/reaction when compared to NTC. n = 8 each circle represents a replicate; *** indicates p ≤ 0.001; **** indicates p ≤ 0.0001.

Figure 3

LAMP heating using a low-cost 3D printer (Monoprice IIIP). (A) 3D printer setup for LAMP incubation. Samples were either placed directly on the heater or in an aluminum block that was on the heated bed. (B) Photos were taken at different time intervals to show the color change over time in positive samples.

Figure 4

Incubation of LAMP reactions using an inexpensive thermos cup. (A) The thermos filled with 65 °C water. (B) Sample tubes were loaded into the sponge so they were in contact with the hot water and afterwards, the thermos lid was sealed to prevent evaporation. (C) End-point image of the reactions. Samples with no template or low concentrations of target DNA remained pink while the positive control (PC) and samples with higher amounts of template turned yellow.