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. Quantitative PCR (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 expand 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 1 hour 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 and is compatible with a low-cost CTAB-based DNA extraction method. 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 DNA extraction method and LAMP assay demonstrate potential for diagnosing Chagas disease in resource-limited endemic regions, particularly where qPCR is unavailable.

Importance: Chagas disease, caused by the parasite Trypanosoma cruzi, is a life-threatening illness that disproportionately affects resource-limited communities. Congenital Chagas disease, if diagnosed early, presents a unique opportunity for intervention, as treatment in newborns is highly effective with minimal side effects. However, early diagnosis is hindered by the high cost and limited availability of current molecular diagnostic methods in endemic regions. Our study introduces a simple, low-cost, and highly specific LAMP assay targeting the HSP70 gene of T. cruzi. This assay is user-friendly, stable under varying storage and incubation conditions, and designed for accessibility in underserved areas. By providing a detailed, open-access protocol and primers, we aim to facilitate the widespread adoption of this diagnostic assay, enabling earlier detection and treatment. This assay lays the groundwork for a new approach to Chagas disease management, potentially reducing the spread of Chagas disease and improving public health outcomes in vulnerable populations globally.