Combination of ultra-rapid DNA purification (PURE) and loop-mediated isothermal amplification (LAMP) for rapid detection of Trypanosoma cruzi DNA in dried blood spots

Abstract

Background Chagas disease or American trypanosomiasis, a neglected tropical disease, is a persistent Public Health problem in Latin America and other, non-endemic, countries. Point-of-care (POC) sensitive methods are still needed to improve and extend early diagnosis in acute infections such as congenital Chagas disease. The objective of this study was to analytically evaluate in the lab the performance of a qualitative POC molecular test (Loop-mediated isothermal amplification (LAMP), Eiken, Japan) for rapid diagnosis of congenital Chagas disease employing FTA cards or Whatman 903 filter paper as solid supports for small-scale volumes of human blood. Methodology/principal findings We used human blood samples artificially infected with cultured T. cruzi strains to assess the analytical performance of the test in comparison with liquid blood anticoagulated with heparin. The DNA extraction process was evaluated using the ultrarapid purification system PURE manufactured by Eiken Chemical Company (Tokio, Japan) over artificially infected liquid blood or different amounts of dried blood spot (DBS) 3- and 6-mm pieces of FTA and Whatman 903 paper. LAMP was performed on a AccuBlock (LabNet, USA) heater or in the Loopamp LF-160 incubator (Eiken, Japan), and visualization of results was either done at naked eye, using the LF-160 device or P51 Molecular Fluorescence Viewer (minipcr bio, USA). Best conditions tested showed a limit of detection (LoD) with 95% accuracy (19/20 replicates) of 5 and 20 parasites/mL, respectively for heparinized fluid blood or DBS samples. FTA cards showed better specificity than Whatman 903 filter paper. Conclusions/significance Procedures to operate LAMP reactions from small volumes of fluid blood or DBS in FTA were standardized for LAMP detection of T. cruzi DNA. Our results encourage prospective studies in neonates born to seropositive women or oral Chagas disease outbreaks to operationally evaluate the method in the field.

In vitro diagnostic methods of Chagas disease in the clinical laboratory: a scoping review

“…In comparison to qPCR for chronic Chagas disease, LAMP appears to be more sensitive ( Besuschio et al, 2017 ). Among other uses, LAMP accuracy using FTA cards compared to heparinized blood was 95% ( Longhi et al, 2023 ), however, no performance analyses were performed.…”

Nucleic acid degradation after long‐term dried blood spot storage

“…1000 bp) can be preserved and amplified even for up to 11 years storage at ambient temperature in hospital settings of tropical regions, but amplification efficacy decreased when samples were stored longer than 9 years (Chaisomchit et al., 2005). PCR‐based target detection performed differently on DNA isolated different preservation papers (Bezerra et al., 2021), in some cases with improved sensitivity when FTA cards were used (Hashimoto et al., 2019; Longhi et al., 2023; Tam et al., 2016). In the current study, the DNA integrity of DBS deteriorated significantly compared with that of cold‐chain stored samples, but retained at a maximum of 20,000 bp fragments regardless of the storage period and filter paper used.…”

“…Multiple types of specialized filter paper have been designed to optimize the collection and stability of biological materials of interest for a variety of research purposes, such as Whatman 903 protein saver, Flinders Technology Associates (FTA) Cards, and Nucleic‐Card (TM) (Marchand et al., 2021; Molteni et al., 2013; Uttayamakul et al., 2005). FTA cards are the best‐established option in nucleic acid‐targeted research (Cardona‐Ospina et al., 2019; da Cunha Santos, 2018; Hashimoto et al., 2019; Longhi et al., 2023; Rahikainen et al., 2016; Tam et al., 2016). FTA cards rely on a porous matrix of cellulose fibre to trap nucleic acid molecules, while they are additionally impregnated with proprietary chemicals that can denature proteins and reduce microbial growth, preventing enzymatic degradation of nucleic acids.…”

Use of sera cell free DNA (cfDNA) and exovesicle-DNA for the molecular diagnosis of chronic Chagas disease

“…CCD poses the greatest epidemiologic risk of disease transmission in countries where infection by insect vectors does not occur, either as a consequence of blood transfusion or transplantation, or as a consequence of transplacental transmission from mother to fetus. Due to the low parasitemia in blood, detection of the parasite is very difficult and new techniques and/or molecular markers are being developed to demonstrate the presence of the parasites or secreted products that indicate their active presence [16][17][18][19][20].…”

“…These methods aim to overcome the problems inherent in the disease itself or in the purification of DNA from blood samples. Thus, the use of chaotropic agents such as guanidine hydrochloride, which denature the proteins in the blood sample prior to DNA purification, new DNA extraction systems [17] and the use of specific primers that increase sensitivity [34,35], especially for those hospital centers where the extraction of nucleic acids is carried out with automatic equipment and in which the extraction efficiency is not the most effective, this together with the development of PCR methodologies to increase sensitivity such as quantitative PCR (qPCR) [18] capable of detecting low concentrations of DNA (0.01 parasites in the sample), have gained ground. Also new amplification systems such as isothermal amplification (LAMP) [17,36] that facilitate the implementation of molecular diagnosis are used.…”