A specific, stable, and accessible LAMP assay targeting the HSP70 gene of Trypanosoma cruzi

Abstract

Diagnostic delays prevent most Chagas disease patients from receiving timely therapy during the acute phase when treatment is effective. qPCR-based diagnostic methods provide high sensitivity during this phase but require specialized equipment and complex protocols. More simple and cost-effective tools are urgently needed to optimize early Chagas disease diagnosis in low-income endemic regions. Here, we present a loop-mediated isothermal amplification (LAMP) that targets a highly conserved region in the HSP70 gene of Trypanosoma cruzi, the causative agent of Chagas disease. This assay demonstrates species-specific amplification across multiple parasite genetic lineages while maintaining stability after 2 hours of incubation and at least 8 months of storage at -20°C. Moreover, the assay is at least 12 times less expensive than the TaqMan qPCR that is currently routinely used for acute Chagas diagnostics. Population-based validation in 100 infants born to Chagas-positive mothers in Santa Cruz, Bolivia, yielded a specificity of 100% and sensitivity exceeding 77% when compared to a TaqMan qPCR that targets satellite DNA. This cost-effective assay holds promise for large-scale diagnosis of Chagas disease in endemic regions with limited resources.

Keywords: Acute Chagas; HSP70 gene; LAMP; congenital transmission; cost-effective method; molecular diagnosis.

Figure 1.. The HSP70 sequence is a molecular marker that differentiates T. cruzi from closely related trypanosomatids.

Neighbor-joining tree analysis based on HSP70 sequences discriminates Leishmania spp (blue), T. rageli (purple), and T. cruzi (green). The horizontal scale represents the evolutionary distance. A scale of 0.03 means an average of 3 substitutions per 100 nucleotides.

Figure 2.. Primers for LAMP_TcHSP70 align to regions that distinguish T. cruzi from Leishmania and T. rangeli.

The HSP70 sequences from twelve representative strains of Leishmania, T. rangeli, and T. cruzi were aligned using the Clustal Omega algorithm. The regions where the primers align in forward and reverse are shown as purple and orange bars, respectively. The inner primers (FIP and BIP) align to two distinct regions, connected by red lines. Nucleotide variants are depicted as small colored bars over the sequences represented as horizontal grey bars. All T. cruzi sequences, except T. cruzi marinkellei, which does not infect humans, show 100% identity in the primer-targeted regions. The figure was generated using Geneious Prime software. Sequences named on the left: 1) Leishmania braziliensis, 2) T. rangeli, 3) T. cruzi marinkellei, 4) T. cruzi Dm28c, 5) T. cruzi G, 6) T. cruzi Sylvio, 7) T. cruzi YC6, 8) T. cruzi Berenice, 9) T. cruzi Brasil A4, 10) T. cruzi TCC, 11) T. cruzi Tulahuen, 12) T. cruzi CL.

Figure 3.. LAMP_TcHSP70 amplifies T. cruzi DNA.

A) Workflow for LAMP_TcHSP70 amplification. B) Visualization of the amplified products by visible light and UV light. 1ng DNA was loaded per sample. C) Visualization of the amplified products by electrophoresis. Representative agarose gel of three replicates showing the classic pattern of LAMP product. 3 μL of the amplified products were visualized on a 2% agarose gel pre-stained with 0.2 μg/mL ethidium bromide. A 100 bp DNA ladder was used as a reference for determining amplicon sizes.

Figure 4.

LAMP_TcHSP70 shows high specificity and sensitivity using parasite-derived DNA. 40 samples were run in parallel. Representative captures of two replicates evaluated by visible and UV light. For all samples, 1 ng of purified DNA was seeded.

Figure 5.. The LAMP_TcHSP70 assay shows a consistent LoD across multiple strains and DTUs, with a LoD in the order of picograms.

A) LoD after the first round of serial DNA dilutions using a factor of 10. B) LoD after the first round of serial DNA dilutions using a factor of 2. C) Design of CRIPRcas12a system. D) LoD ofter using LAMP_TcHSP70 coupled to CRIPRcas12a system. Three independent replicates per strain. Mean fluorescence intensity was estimated using the ROI manager tool of Fiji 2 software. A-B) Error bars indicate standard error. The horizontal red line highlights the mean fluorescence intensity reached by the negative control.

Figure 6.. The LAMP targeting T.cruzi HSP70 remains stable even after extended incubation and storage times.

A) Extended incubation times do not reveal non-specific amplification in negative controls. Data represent four technical replicates per incubation period. Human DNA (negative control) was seeded at 50 ng, while T. cruzi DNA was seeded at 2 pg. B) Master mix storing for eight months does not affect LAMP performance. Data represent three technical replicates per storing period. One reaction was used for negative control. Human DNA (negative control) was seeded at 50 ng, while T. cruzi DNA was seeded at 1 ng. Error bars indicate standard error. Mean fluorescence intensity was estimated by ROI area using Fiji software.

Figure 7.

The LAMP_TcHsp70 assay exhibits high specificity and accuracy but lower sensitivity compared to SatDNA-qPCR. A) Sensitivity, specificity, and accuracy per test. B) Sensitivity, specificity, and accuracy per patient diagnosis. C) Positive and negative LAMP_TcHSP70 results in relation to parasitic load (parasites/mL) as estimated by the qPCR calibration curve. D) Alignment of HSP70 sequences from clinical isolates.