Toward the Establishment of a Single Standard Curve for Quantification of Trypanosoma cruzi Natural Populations Using a Synthetic Satellite Unit DNA Sequence

Abstract

Accurate diagnostic tools and surrogate markers of parasitologic response to treatment are needed for managing Chagas disease. Quantitative real-time PCR (qPCR) is used for treatment monitoring, but variability in copy dosage and sequences of molecular target genes among different Trypanosoma cruzi strains limit the precision of quantitative measures. To improve qPCR quantification accuracy, we designed and evaluated a synthetic DNA molecule containing a satellite DNA (satDNA) repeat unit as standard for quantification of T. cruzi loads in clinical samples, independently of the parasite strain. Probit regression analysis established for Dm28c (TcI) and CL-Brener (TcVI) stocks similar 95% limit of detection values [0.903 (0.745 to 1.497) and 0.667 (CI, 0.113 to 3.927) copy numbers/mL, respectively] when synthetic DNA was the standard for quantification, allowing direct comparison of loads in samples infected with different discrete typing units. This standard curve was evaluated in 205 samples (38 acute oral and 19 chronic Chagas disease patients) from different geographical areas infected with various genotypes, including samples obtained during treatment follow-up; high agreement with parasitic load trends using standard curves based on DNA extracted from spiked blood with counted parasites was obtained. This qPCR-based quantification strategy will be a valuable tool in phase 3 clinical trials, to follow up patients under treatment or at risk of reactivation, and in experimental models using different parasite strains.

Parasitemia Levels in Trypanosoma cruzi Infection in Spain, an Area Where the Disease Is Not Endemic: Trends by Different Molecular Approaches

“…As we do not know the real diversity of the T. cruzi populations affecting each patient, selecting the predominant DTU to build the standard curve does not seem to be the best option if we want to compare data from different regions with different circulating DTUs. To overcome this situation, the adoption of a single standard curve for the quantification of T. cruzi using a synthetic satellite unit DNA sequence was recently proposed ( 27 ). However, this strategy is also unable to estimate the parasitic load due to the impossibility of converting Sat-DNA copies into numbers of parasites without knowing which DTUs are infecting each patient.…”

“…Therefore, a consensus on the best strategy to build a standard curve is needed. A potentially suitable candidate could be TcI because it has the lowest Sat-DNA copy number ( 27 ), it is the most ubiquitous DTU in areas of endemicity, and patients with TcI infection have been reported from North, Central, and South America ( 28 , 29 ).…”

Validation of the NAT Chagas IVD Kit for the Detection and Quantification of Trypanosoma cruzi in Blood Samples of Patients with Chagas Disease

“…For the NAT Chagas kit, two positive controls (synthetic double-stranded T. cruzi satDNA conserved sequence, at 100 and 10 copies/μL), supplied with the kit, were used in each assay. To obtain the 166-bp sequence for the synthetic DNA controls, the satDNA repeats sequences from strains and clones representing T. cruzi DTUs I to VI available at the GenBank were aligned, showing the high conservation of the repeats [ 39 ]. As an exogenous control, a 181-bp double-stranded synthetic DNA sequence, also supplied with the kit, was used, as described in the DNA extraction section.…”

“…Eq./mL. (b) Synthetic satDNA: the second standard curve was prepared from synthetic T. cruzi satDNA double-strand template, as reported in [ 39 ]. Briefly, DNA obtained from GEB-seronegative samples was mixed with the synthetic T. cruzi satDNA template at 10 8 copies/mL, and 1/10 serial dilutions were performed as described for the standard curve generated by DNA extracted from T. cruzi parasites, ranging from 10 5 to 1 satDNA copies/mL.…”

Synthetic oligonucleotides as quantitative PCR standards for quantifying microbial genes

“…The synthetic gene fragments can be purchased in a length of 125 to 3,000 base pair (bp) with known degenerate nucleotides of A, T, C, and G ( May et al, 2015 ; Conte et al, 2018 ). Up to now, most of the artificially synthesized standards have been used for medical purpose, focusing on viral or infectious microorganisms ( Tourinho et al, 2015 ; Fesolovich and Tobe, 2017 ; Lima et al, 2017 ; Magee et al, 2017 ; Bandeira et al, 2020 ; Bivins et al, 2021 ; Munoz-Calderon et al, 2021 ), very few in environmental samples. To the best of our knowledge, the few studies using synthesized gene fragments as qPCR standards in environmental microbiology studies assessed bacterial 16S rRNA and htrA genes in soils ( Gunawardana et al, 2014 ; Sirois and Buckley, 2019 ), 16S rRNA genes and methanogenic mcr A in a biogas digester ( May et al, 2015 ), antibiotic resistance genes in environmental water, soil and faeces samples ( Xu et al, 2019 ), and 16S rRNA genes by adding synthetic DNA internal standard to fecal samples ( Zemb et al, 2020 ).…”