Development of an Aptamer-Based Concentration Method for the Detection of Trypanosoma cruzi in Blood

Abstract

Trypanosoma cruzi, a blood-borne parasite, is the etiological agent of Chagas disease. T. cruzi trypomastigotes, the infectious life cycle stage, can be detected in blood of infected individuals using PCR-based methods. However, soon after a natural infection, or during the chronic phase of Chagas disease, the number of parasites in blood may be very low and thus difficult to detect by PCR. To facilitate PCR-based detection methods, a parasite concentration approach was explored. A whole cell SELEX strategy was utilized to develop serum stable RNA aptamers that bind to live T. cruzi trypomastigotes. These aptamers bound to the parasite with high affinities (8–25 nM range). The highest affinity aptamer, Apt68, also demonstrated high specificity as it did not interact with the insect stage epimastigotes of T. cruzi nor with other related trypanosomatid parasites, L. donovani and T. brucei, suggesting that the target of Apt68 was expressed only on T. cruzi trypomastigotes. Biotinylated Apt68, immobilized on a solid phase, was able to capture live parasites. These captured parasites were visible microscopically, as large motile aggregates, formed when the aptamer coated paramagnetic beads bound to the surface of the trypomastigotes. Additionally, Apt68 was also able to capture and aggregate trypomastigotes from several isolates of the two major genotypes of the parasite. Using a magnet, these parasite-bead aggregates could be purified from parasite-spiked whole blood samples, even at concentrations as low as 5 parasites in 15 ml of whole blood, as detected by a real-time PCR assay. Our results show that aptamers can be used as pathogen specific ligands to capture and facilitate PCR-based detection of T. cruzi in blood.

Chagas Disease Diagnostic Applications

“…Later on, a large repertoire of kDNA or nuclear DNA targets including single- or multi-copy genes and satellite sequences, as well as different multi-target strategies have been explored (Schijman et al, 2011). More recently, a parasite concentration approach based on short and stable RNA aptamers was developed to facilitate PCR-based detection methods, and proposed as a potential alternative tool in monitoring parasite load in Chagasic patients (Nagarkatti et al, 2012; Nagarkatti et al, 2014). …”

Putting Infection Dynamics at the Heart of Chagas Disease

“…Emerging technologies could soon increase sensitivity further and generate new insights into the parasite’s biology in vivo. For example, aptamers [35] or spliced leader trapping [36] can potentially enrich samples for rare parasites or parasite-derived mRNAs, while ribosomal profiling [37], fluorescence dilution [38] and isotope labelling [39] approaches could help differentiate between dormant and active parasites. In the following sections we review current understanding of T. cruzi infection dynamics and explore why the utility of the parasite detection techniques described above depends on appropriate tissue sampling strategies.…”

The use of laser‐based diagnostics for the rapid identification of infectious agents in human blood

“…The extracellular trypomastigotes of the parasite, T. cruzi (Tulahuen strain), were cultured in 3T3 cells as previously described (Nagarkatti et al . ) and quantified with a cellometer (Vision, Nexcelom Bioscience, Lawrence, MA) and by real‐time PCR as previously described (Kourout et al . ).…”

“…Concentrations in the spiked specimens were used without further verification. The extracellular trypomastigotes of the parasite, T. cruzi (Tulahuen strain), were cultured in 3T3 cells as previously described (Nagarkatti et al 2012) and quantified with a cellometer (Vision, Nexcelom Bioscience, Lawrence, MA) and by real-time PCR as previously described (Kourout et al 2016). Spiking of S. aureus, Y. pseudotuberculosis, HIV-1 (HIV) and T. cruzi was performed as previously described (Grigorenko et al 2017).…”