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.

Trypanosoma cruzi chromosome ends are enriched in surface protein genes and pseudogenes (e.g., trans-sialidases) surrounded by repetitive sequences. It has been proposed that the extensive sequence variability among members of these protein families could play a role in parasite infectivity and evasion of host immune response. In previous reports we showed evidence suggesting that sequences located in these regions are subjected to recombination. To support this hypothesis we introduced a double-strand break (DSB) at a specific target site in a T. cruzi subtelomeric region cloned into an artificial chromosome (pTAC). This construct was used to transfect T. cruzi epimastigotes expressing the I-SceI meganuclease. Examination of the repaired sequences showed that DNA repair occurred only through homologous recombination (HR) with endogenous subtelomeric sequences. Our findings suggest that DSBs in subtelomeric repetitive sequences followed by HR between them may contribute to increased variability in T. cruzi multigene families.

Trypanosoma cruzi has been subdivided into seven Discrete Typing Units (DTUs), TcI-TcVI and Tcbat. Two major evolutionary models have been proposed to explain the origin of hybrid lineages, but while it is widely accepted that TcV and TcVI are the result of genetic exchange between TcII and TcIII strains, the origin of TcIII and TcIV is still a matter of debate. T. cruzi satellite DNA (SatDNA), comprised of 195 bp units organized in tandem repeats, from both TcV and TcVI stocks were found to have SatDNA copies type TcI and TcII; whereas contradictory results were observed for TcIII stocks and no TcIV sequence has been analyzed yet. Herein, we have gone deeper into this matter analyzing 335 distinct SatDNA sequences from 19 T. cruzi stocks representative of DTUs TcI-TcVI for phylogenetic inference. Bayesian phylogenetic tree showed that all sequences were grouped in three major clusters, which corresponded to sequences from DTUs TcI/III, TcII and TcIV; whereas TcV and TcVI stocks had two sets of sequences distributed into TcI/III and TcII clusters. As expected, the lowest genetic distances were found between TcI and TcIII, and between TcV and TcVI sequences; whereas the highest ones were observed between TcII and TcI/III, and among TcIV sequences and those from the remaining DTUs. In addition, signature patterns associated to specific T. cruzi lineages were identified and new primers that improved SatDNA-based qPCR sensitivity were designed. Our findings support the theory that TcIII is not the result of a hybridization event between TcI and TcII, and that TcIV had an independent origin from the other DTUs, contributing to clarifying the evolutionary history of T. cruzi lineages. Moreover, this work opens the possibility of typing samples from Chagas disease patients with low parasitic loads and improving molecular diagnostic methods of T. cruzi infection based on SatDNA sequence amplification.