Chagas disease, caused by the intracellular parasite Trypanosoma cruzi, is one of the most important parasitological infections in the Americas. It is estimated to infect approximately 6 million people from mostly low income countries in Latin America, although recent infections have been reported in southern US states. Several studies have described an extensive genetic diversity among T. cruzi isolates throughout its geographic distribution in the American continent. This diversity has been correlated with the pathology developed during an infection. However, due to a lack of a single reliable test, current diagnosis practices of the disease are not straightforward since several different tests are applied. The use of current genomic sequence data allows for the selection of molecular markers (MM) that have the ability to identify the Discrete Typing Unit (DTU) of T. cruzi in a given infection, without the need of any sequencing reaction.
Applying three criteria on the genomic sequencing data of four different phylogenetic lineages of T. cruzi, we designed several molecular tests that can be used for the molecular typing of the parasite. The criteria used were: (1) single-copy orthologs of T. cruzi, (2) T. cruzi unique loci, and (3) T. cruzi polymorphic loci. All criteria combined allowed for the selection of 15 MM, 12 of which were confirmed to be functional and replicable in the laboratory with sylvatic samples. Furthermore, one MM produced distinct polymerase chain reaction (PCR) amplicon sizes among distinct T. cruzi DTUs, allowing the use of a AFLP-PCR test to distinguish DTUs I, II/IV, V and VI. Whereas two MM can differentiate DTUs I, II, IV and V/VI out of the six current DTUs with a PCR-RFLP test.
The designed molecular tests provide a practical and inexpensive molecular typing test for the majority of DTUs of T. cruzi, excluding the need to perform any sequencing reaction. This provides the scientific community with an additional specific, quick and inexpensive test that can enhance the understanding of the correlation between the DTU of T. cruzi and the pathology developed during the infection.
Citation: Rivas-García L, Carballo-Amador MA, Flores-López CA (2020) Design of a AFLP-PCR and PCR-RFLP test that identify the majority of discrete typing units of Trypanosoma cruzi. PLoS ONE 15(8): e0237180. https://doi.org/10.1371/journal.pone.0237180
Editor: Ruslan Kalendar, University of Helsinki, FINLAND
Received: May 8, 2020; Accepted: July 21, 2020; Published: August 4, 2020
Copyright: © 2020 Rivas-García et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: CAFL: received a grant of Problemas Nacionales, CONACYT, Mexico. Grant number: 2015-01-547. Funder full name: Consejo Nacional de Ciencia y Tecnología URL: https://www.conacyt.gob.mx/ The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Trypanosoma cruzi is the etiological agent of American trypanosomiasis, also known as Chagas disease . No vaccine has been developed for this infection, and the two drugs available to treat it are of limited use and may present severe secondary effects . The pathology associated with the infection consists of two stages: an acute phase (the first 2–3 weeks of infection) characterized by high parasitemia and a chronic phase (10–30 years after infection) defined by low parasitemia and syndromes mostly associated with heart failure, megacolon, and megaesophagus . The disease is mostly considered to be endemic to Latin America, where the parasite is usually transmitted by its vector-mediated transmission or via the oral path of transmission . However, recent studies have found autochthonous cases of infection in the southeastern United States [3, 4]. The disease is currently estimated to affect approximately 6 million people . However, this number is challenging to assess, given the limited amount of data, inefficient public health systems.
Currently, the World Health Organization (WHO) considers direct parasitological tests of blood smears for the acute phase and any two different serologic tests (enzyme-linked immunosorbent assay [ELISA], indirect immunofluorescence, and/or indirect hemagglutination) for the chronic phase to be the standard diagnostic practices for the infection, as a result of the lack of a gold-standard test [5, 6]. However, several alternative diagnostic procedures have been developed (xenodiagnosis, blood smears, strout, microstrout, microhematocrit, hemoculture, PCR, and quantitative PCR [qPCR]) , since the standard diagnosis continues to be inaccurate. Some of these are considered “indirect” tests because they do not directly detect the presence of the parasite, but rather detect the presence of antibodies, which cannot discriminate among cases where the host immune system might have neutralized the pathogen. The development of “direct” diagnostic tests, like a PCR targeted at amplifying T. cruzi unique loci, has been prioritized [8, 9].
In addition to the nature of the test, the genetic background of the parasite needs to be considered. The genetic diversity of this mostly asexual parasite is classified into what is known as discrete typing units (DTUs), labeled I to VI, with a recent seventh genetic lineage termed TcBat [10–12]. However, stringent phylogenetic analyses can reduce this classification system into three or four monophyletic clades, depending on the MM used (three with mitochondrial loci and four with nuclear loci) (S1 Table) [13, 14]. To date, three of the four monophyletic clades are represented in the annotated genomes sequenced [15–17]. There can be as much as a 20–40% difference in genome content among some phylogenetic groups , which highlights the potential biological differences among the different T. cruzi DTUs presently circulating in nature.
The characterization of this parasite’s diversity is essential, since a correlation of the pathology of the infection with the genetic background of the parasite has been reported [18–23]. Although other factors appear to play a role in the severity of a given infection , identifying the given genetic background of the parasite in a given human infection is crucial for further understanding of the disease.
The potential medical benefit attained from sequencing genomes of human pathogens cannot be undervalued. For T. cruzi, the first genomic sequence became available almost 15 years ago , marking an enormous accomplishment for neglected tropical diseases. A project that was specially challenging to accomplish due to the vast diversity of surface protein coding genes intrinsic to T. cruzi genomes. Fortunately, currently there are several annotated genomes of T. cruzi, representing a significant percentage of the total genomic diversity described so far . It is vitally significant to identify molecular markers with the ability to distinguish the genetic background of a pathogen of interest in the absence of DNA sequencing.
To accomplish this objective, three criteria were targeted for the design of the molecular tests: single-copy orthologs, T. cruzi species-specific loci, and genetically diverse loci. The rationales behind each of these criteria were as follows: (1) selecting single-copy orthologs would prevent the amplification of paralogs, since the use of paralogs has the potential of amplifying multiple amplicons due to mispriming during the PCR; (2) selecting loci unique to T. cruzi would ensure a species-specific molecular typing test for the infection by T. cruzi; and finally, (3) selecting genetically diverse loci would allow the identification of the DTUs of T. cruzi by either distinctive PCR amplicon sizes or the development of unique restriction fragment length digestion profiles. This would potentially produce MM with the power to differentiate the major DTUs of T. cruzi without a need to sequence the amplicons, once all three criteria are combined. Resulting in an alternative molecular typing test that is quicker and independent to a Sanger sequencing based reaction, which although is comparable in cost, is not necessarily a readily available option in most Latin American laboratories.
Materials and methods
Design of molecular tests
To select the loci with the traits required for the molecular typing test to be species specific (i.e., to amplify solely in T. cruzi) and accurate (i.e., to be able to differentiate the majority of DTUs of the parasite), genome sequences of the four annotated T. cruzi strains available in February 2017 at TriTrypDB were used . The strains analyzed were Sylvio classified as DTU I , Dm28c cataloged as DTU I , Esmeraldo classified as DTU II , and CL Brener classified as DTU VI .
Selection of single-copy orthologs was accomplished by excluding paralogs from the selection. Any self-BLAST hits with an E value <0.05 were excluded . Selection of loci unique to T. cruzi was attained by confirming the uniqueness of the loci for T. cruzi in Genbank’s nr/nt database  and by excluding any loci that had another BLAST E value hit of <0.001 in any other taxonomic unit. To avoid mispriming to other non T. cruzi species, the number of SNPs found within the primer annealing regions were quantified by comparing these target sequences among the Trypanosoma genus sequence data. Finally, for the selection of genetically diverse loci, ortholog sequences from the four genomes used were aligned and orthologs were prioritized according to their diversity in terms of distinct gene sizes among DTUs and/or the highest number of SNPs among DTUs.
Primer design and PCR
Orthologs that fulfilled all of the three criteria previously mentioned were selected as potential loci for primer design that would target their amplification via PCR. Primers were designed in Primer3 , where the annealing position was selected while trying to avoid polymorphic regions, hence increasing the likelihood of the primers annealing on all of the T. cruzi genetic groups. Thus, we focused on heavily conserved regions within the loci for their design, based on the sequence alignments of the four phylogenetic lineages available. In addition, the design of relatively small amplicons was favored, without sacrificing the genetic diversity recovered. This favors efficient PCR amplification from samples with poor DNA quantity, a scenario commonly encountered in chronic Chagas infections.
The PCR optimization reactions were assayed under the following PCR amplification conditions: an initial denaturation step at 95°C for 4 min, followed by 35 cycles of a 30-s denaturation step at 95°C, annealing temperature with a gradient of ±2°C, focused on the average annealing temperature for each set of primers for 50 s, extension at 72°C for 50 s, and a final extension of 5 min at 72°C. PCR was performed in a Veriti 96-Well Fast Thermal Cycler from Applied Biosystems. PCR reactions had the following volumes: 1.5 μL of 10× PCR Rxn Buffer (-MgCl2), 0.45 μL MgCl2 (50 mM), 0.3 μL dNTPs (10 mM each) (VWR Life Science Amresco), 0.75 μL for each primer (50 pmol), 0.06 μL Taq polymerase (5 U/μL) (Invitrogen), 1 μL of DNA (10 ng/μL), and DEPC water to obtain a final volume of 15 μL per sample.
The PCR amplicons were confirmed by agarose 1% (w/v) gel electrophoresis at 80 V for 30 min in a mini sub cell (BioRad) stained with SYBR Safe DNA gel stain (Invitrogen, USA). DNA fragments were visualized under ultraviolet (UV) light with the Azure c200 gel documentation platform. The PCR performance was tested using T. cruzi clone DNA samples representing all major DTUs (except for DTU III), which were kindly provided by Dr. Carlos Machado at the University of Maryland. Once the functionality of the PCR was established, DNA extracted from the digestive tracts of field collected Dipetalogaster maxima individuals were used. These samples were part of an independent study aimed at describing the natural infection rate of T. cruzi in this Baja California endemic species throughout a three-year study period (unpublished results). In this study the natural infection rate was determined by using two distinct T. cruzi specific MM (COII-NDI & Tc00.1047053506529.310) [13, 14]. Posteriorly, a random set of four positive T. cruzi DNA samples from this study were used to verify the efficiency of the MM.
The species specificity of the MM was tested on DNA of five distantly related taxonomical orders (Artiodactyla, Asterales, Rhabditida, Chytridiales, and Coleoptera). To further evaluate the specificity of the primers, the SNPs present within the annealing regions of all primers among other Trypanosoma spp. were quantified (for T. rangeli, T. vivax, T. evansi, T. theileri, and T. cruzi marinkellei).
Discrete typing unit discrimination
Genetic loci that would produce distinct PCR amplicon sizes for each major phylogenetic group were selected as potential MM. Likewise, the polymorphic sites detected by aligning the homolog sequences from all selected T. cruzi loci were designated as potential targets to discover restriction enzymes that would selectively produce distinct length profiles via a PCR restriction fragment length polymorphism (RFLP) test. These targets were analyzed in Serial Cloner 2.6.1 to screen for potential restriction endonucleases that would differentiate among the major DTUs in each infection. The enzymatic reactions were performed according to the manufacturer’s protocol (New England BioLabs), using the amplicons of distinct T. cruzi DTUs as a template (S1 Table). The T. cruzi strains for which a DNA template was used in PCR (S1 Table) were distinct from the T. cruzi strains for which genomes were used in the primer design procedure. Hence, the PCR amplicons used for the PCR-RFLP were sequenced prior to their respective enzymatic digestion, to evaluate the conservation of restriction digestion sites among distinct strains classified within the same DTU. This was a necessary appraisal that provided only a little information into the conservation of SNPs, since very few strains of T. cruzi have had their respective genomes sequenced, severely limiting the assessment of the degree of conservation of the restriction digestion sites among the DTUs. Samples were analyzed by electrophoresis in a 3% (w/v) agarose gel at 110 V for 50 min in a mini sub cell stained with SYBR Safe DNA gel stain and were visualized with Azure c200 platform.
For the amplified fragment length polymorphism (AFLP) PCR test, digested samples were separated by native polyacrylamide gel electrophoresis with 10% (w/v) acrylamide using the Mini-PROTEAN Tetra Cell (BioRad). Samples containing equal volumes (10 μL) of PCR product were mixed with 6× Novel Juice loading dye (Sigma-Aldrich) and separated in a 1.0-mm-thick minigel (3.9 mL water, 0.6 mL 5× TBE, 1.5 mL 38:2 acrylamide:bis, 22.5 μL 10% [w/v] APS, 7.5 μL TEMED) in 0.5× TBE buffer at 50 V for 10 min and then increased to 100 V for 60 min (modified from ). Bands were visualized with the Azure c200 platform.
Primer design, PCR, and multiplex
A total of 13 loci met the criteria established in our experimental design. In terms of potential gene candidates, the most stringent criterion was selecting single-copy orthologs. Applying all three criteria of our experimental design (single-copy genes, genetically diverse loci and T. cruzi species-specific loci) resulted in a total of 15 gene candidates, however two of these were not suitable for proper oligonucleotide design. This resulted in the selection of 15 MM (two loci had two distinct molecular tests designed for each loci). With regard to the function of the 13 candidate loci selected, some had a relationship to catalytic functions, such as TcSC5D, which is another T. cruzi MM that was developed in 2012 for the diagnosis of the disease . In addition, there were loci involved in metabolic pathways for the biosynthesis of amino acids, membrane lipids and glycine, among other functions.
The optimization of the primers designed resulted in only 12 MMs amplifying successfully, confirming their potential use in the molecular typing of the majority DTUs of the parasite (Table 1 and Fig 1).
Lane number: (1) strain Tulacl2 (DTU VI); (2) strain PSC-O (DTU V); (3) strain EV13c (DTU I); (4) strain CANIII (DTU IV); (5) strain CA-1-05 (DTU I); (6) strain CBB cl3 (DTU II); (7) strain ESMclZ2 (DTU II). Negative control (C(-)). DNA ladder (L) (Fermentas, #SM1163).
Discrete typing unit identification
The SNPs identified as potential restriction sites were used to select potential restriction enzymes that could differentiate among the major DTUs representing the known genetic diversity of the parasite. A total of eight restriction enzymes were found to be appropriate (S2 Table). Among the 12 effective MM, only the hypothetical protein (TcCLB.506575.9) had distinct gene sizes between the 3 DTUs compared. Two MMs had restriction sites appropriate for the differentiation of four DTUs of the parasite (ccr4-not transcription complex subunit (Tc00.1047053510535.60) and diacylglycerol acyltransferase (TcCLB.507467.90)), while one MM could only discriminate DTU II (amino acid permease-like protein (Tc00.1047053510251.10)) (Table 2). The theoretical digested fragment sizes were previsualized in Serial Cloner 2.6.1. and were corroborated in the lab (Fig 2) using DNA from T. cruzi clones kindly provided by Dr. Carlos Machado at the University of Maryland (S1 Table).
Panel A: diacylglycerol acyltransferase (TcCLB.507467.90) digested with SalI. Panel B: diacylglycerol acyltransferase (TcCLB.507467.90) digested with AluI. Panel C: ccr4-not transcription complex subunit (Tc00.1047053510535.60) digested with BseYI. Panel D: ccr4-not transcription complex subunit (Tc00.1047053510535.60) digested with AvalI. Panel E: amino acid permease-like protein (Tc00.1047053510251.10) digested with FauI. Panel F: amino acid permease-like protein (Tc00.1047053510251.10) digested with BstZ17l. Lane numbers for panels A-D and F: (1) strain EV-13C (DTU I); (2) strain CA-1-05 (DTU I); (3) strain CBBcl3 (DTU II); (4) strain ESMcl3Z2 (DTU II); (5) strain CANIII (DTU IV); (6) strain PSC-O (DTU V); (7) strain Tulacl2 (DTU VI). Lane numbers for panel E: (1) strain Ev13C (DTU I); (2) strain ESMcl3Z2 (DTU II); (3) strain CANIII (DTU IV); (4) strain PSC-O (DTU V); (5) strain Tula cl2 (DTU VI). DNA ladder (L) (NEBioLabs, #N0551S).
Both the ccr4-not transcription complex subunit (Tc00.1047053510535.60) and the F1/R1 diacylglycerol acyltransferase (TcCLB.507467.90) MM are able to independently differentiate DTU I, DTU II, DTU IV, and DTU V/VI from each other (Table 2, Fig 2A–2D).
The amplified fragment length polymorphism (AFLP-PCR) of the hypothetical protein (TcCLB.506575.9) yields different PCR amplicon sizes for at least four distinct DTUs. This amplified a 377 bp fragment for DTU I, a 397 bp segment for DTUs II and IV, a 405 bp fragment for DTU V, and a 383 bp segment for DTU VI, with an additional nonspecific amplicon of approximately 850 bp for DTU V. These results corroborated the capacity of this particular MM in discriminating four of the six DTUs of T. cruzi without the need to digest the amplicon (Fig 3).
Lane number: (1) strain EV-13C (DTU I), 377 bp amplicon; (2) strain CA-1-05 (DTU I), 377 bp amplicon; (3) strain CBBcl3 (DTU II), 397 bp amplicon; (4) strain ESMcl3Z2 (DTU II), 397 bp amplicon; (5) strain CANIII (DTU IV), 397 bp amplicon; (6) strain PSC-O (DTU V), 405 & 850 bp amplicons; (7) strain Tulacl2 (DTU VI), 383 bp amplicon. DNA ladder (L) (NEBioLabs, #N0551S).
Effectivity in sylvatic samples
To compare the effectiveness of the proposed MM with the previously published ones, a frequently used and cited PCR-based diagnosis was included (TcSC5D gene) [8, 31]. The test was conducted using DNA extracted from T. cruzi clones and field-collected infected bugs (Dipetalogaster maxima). All results (both TcSC5D MM and the newly proposed MM) were positive, which highlighted the effectiveness of these MMs (S1 and S2 Figs).
The results are indicative of a lower PCR yield from DNA extracted from sylvatic specimens versus DNA clones (S1 Fig). This pattern was consistent with the previously published MM . Comparing the PCR yield of a commonly used MM  and the MMs selected in this study, these yields appear comparable, with the exception of a few of the proposed MMs that appear to have higher yields (ammonium transporter (Tc00.1047053508317.50), both diacylglycerol acyltransferase markers (TcCLB.507467.90), the ccr4-not transcription complex subunit (Tc00.1047053510535.60), and the hypothetical protein used as the AFLP-PCR marker (TcCLB.506575.9)) (S1 Fig).
A complete picture of the genetic background of T. cruzi has been partially explored [11, 12], since currently the genomes of a limited number of strains of the parasite have been sequenced and novel genetic diversity from nature continues to be described [11, 32]. A complete representation of the genomic sequences of all DTUs is still lacking, although the genomic data that are currently available for T. cruzi are not insignificant [15–17]. Thus, the annotated genomic sequences of four distinct strains of T. cruzi were used to assist selection of MM with the traits needed to differentiate the DTUs of the parasite. Moreover, multiple thorough phylogenetic analyses condense the current six-DTU classification system employed for T. cruzi into a classification of three to four monophyletic clades [13, 14]. This implies that the DTUs represented in this study cover three of the four monophyletic clades that have thus far been described for this parasite in nature.
Therefore, the initial goal in this study was to find multiple MM that would differ in the PCR amplicon sizes among major DTUs (AFLP-PCR), which would avoid the use of the enzymatic digestion of the PCR amplicon, making the diagnosis faster, easier, and cheaper. This objective was achieved with only the hypothetical protein marker (TcCLB.506575.9). Two additional MMs were selected with the ability to differentiate DTUs I, II, IV and V/VI with an additional RFLP approach.
Applying the most appropriate criteria of traits for our goal led to the selection of the potential loci. The criterion of finding single-copy orthologs was the most stringent. After all our criteria were applied, 13 loci were selected. The protein functions of the selected loci varied. The functions included catalytic enzymes, intracellular transport of amino acids, cell membrane structure, intracellular vesicles, oxidoreductase, ammonium transporter, and a gene with unknown function (Table 1).
PCR-RFLP and AFLP-PCR validation
Of the 15 MMs selected, 12 were successfully amplified in the PCR reaction (Fig 1). Three MMs could not be amplified, which could be due to the following reasons: (A) the presence of SNPs in the annealing regions in the DNA strains used, since not all polymorphisms could be detected in silico, given the actual genetic diversity available, (B) undetected primer dimer formation of the oligonucleotides, and (C) annealing temperatures much lower than those used in the initial primer characterization. In terms of the specificity of the primers, a specificity test (S2 Fig) confirmed the singularity of the MM, specific to the T. cruzi genome. Furthermore, a bioinformatics analysis confirmed the presence of multiple SNPs in the annealing primer regions of closely related species of the genus Trypanosoma (Table 3).
Of the 12 MMs that were successful in their respective PCR amplifications, two had the genomic diversity needed to select restriction enzymes that would produce different digestion patterns for the distinction of four DTUs (I, II, IV and V/VI) out of the six current DTUs (Fig 2A–2D), while 1 MM could only differentiate between two DTUs (Fig 2E and 2F). All MM confirmed the in silico digestion fragment sizes predicted. Furthermore, the efficiency of the PCR amplification was compared to a recent and commonly used PCR MM (S1 Fig) . The results clearly showed that the markers in this study provide an additional typing test for T. cruzi. One of the MMs selected in this study can differentiate, with AFLP-PCR, among four distinct DTUs (Fig 3). The electrophoresis analysis of this MM (hypothetical protein: TcCLB.506575.9) revealed that the PCR is not 100% specific in DTU V, since an additional weak amplicon was observed, most likely due to the presence of a paralogous copy of this gene. This has the potential to increase the diagnostic ability of this particular MM. As a result, we recommend using either the ccr4-not transcription complex subunit (Tc00.1047053510535.60) MM with BseYI and AvaII or the diacylglycerol acyltransferase (TcCLB.507467.90) MM with SalI and AluI (Table 2), since these two markers appear to have the highest resolution of all twelve selected MM.
It should not be surprising that the PCR yield is much lower when the template DNA is extracted from sylvatic specimens, compared to in vitro clones. The lower presence of parasites in sylvatic infections will result in a reduced quantity of template DNA for the PCR. This is especially clear in S2 Fig. In every case (including the control TcSC5D MM and our MMs), the PCR bands that were derived from T. cruzi clone DNA were much stronger than the amplifications using sylvatic DNA templates. This is one of the reasons why many studies usually target a genomic region that might have multiple copies per cell (e.g., kinetoplast chromosomes) [33, 34]. In this study a simple solution could have been to select a paralog gene instead of orthologs. This would have been a suitable approach if the main goal were to simply diagnose the infection, since the larger amount of genetic copies found within the genome would translate into a higher yield from PCR. However, finding a paralog that would produce a digestion pattern unique for each DTU is very unlikely, given the large number of repetitive regions characteristic of T. cruzi .
This study focused on selecting single-copy orthologs as targets. This strategy would ultimately allow the identification of the genetic background of the parasite, which was the ultimate objective of the study. A setback to the study was the lower PCR yield compared to targeting a multiple-copy DNA region.
The ability to identify DTUs of T. cruzi
Since the genetic diversity of T. cruzi is currently categorized in six DTUs and recent studies suggest that there might be an even larger amount of genetic diversity , resolution of the MM in discriminating all DTUs cannot currently be achieved. When a more complete understanding of the real genetic diversity of the parasite has been described, a broader genomic search for better MM will be possible. However, many studies have consistently shown that the genetic diversity of the parasite is not necessarily naturally divided into seven DTUs [13, 14, 35, 36]; it more closely assembles into three or four genetic clusters, depending on whether a mitochondrial or a nuclear marker is used. Further application of this division would reclassify the genetic diversity of the parasite into four or five DTUs (since TcBat has usually not been included in the three or four genetic clade studies), and this in turn would mean these markers could still be valid and able to distinguish types within the main genetic diversity of the parasite.
Genomic sequences were successfully used to design molecular tests with an ability to identify the genetic background of T. cruzi without the need to perform any sequencing reaction. Although the complete genetic diversity of this neglected disease has not been completely explored, the available genomic sequence was vital for the development of novel molecular tests and their potential application for diagnostic purposes. When a more comprehensive description of all the genetic diversity of T. cruzi is accessible, the development of additional and more efficient genetic markers will be possible.
Future work should focus on the application of the MM described in clinical samples. Part of this work is currently being done in collaboration with Centro Regional de Investigación en Salud Pública, Chiapas, México.
S1 Fig. PCR yield in biological versus clone-derived DNA.
Numbers with apostrophe (e.g., 2’) represent PCR amplicons derived from cultured T. cruzi DNA (DTU II strain CBBcl3), whereas numbers without apostrophe represent PCR amplicons derived from DNA extracted from field-collected specimens of Dipetalogaster maxima, previously diagnosed in our lab as positive for T. cruzi. L: DNA ladder (BioTang, UMR-150). Lanes: (1) TcSC5D; (2) amino acid permease-like protein (Tc00.1047053510251.10); (3) ammonium transporter (Tc00.1047053508317.50); (4) CCR 4 (Tc00.1047053510535.60); (5) cystathionine gamma lyase 1 (TCDM_00169 F1 & R1); (6) cystathionine gamma lyase (TCDM_00169 F2&R2); (7) diacylglycerol acyltransferase 1 (TcCLB.507467.90 F1 & R1); (8) diacylglycerol acyltransferase 2 (TcCLB.507467.90 F2 & R2); (9) glycine synthase (BCY84_19826); (10) hypothetical protein (TcCLB.506575.9); (11) kinetoplastid membrane protein (TcSYL_0115580); (12) pyrroline 5 carboxylate reductase (Tc00.1047053506857.20); (13) thioredoxin putative (TcCLB.510227.29).
S2 Fig. Specificity of primers on non-T. cruzi DNA.
Numbers from one to six represent PCR amplicons derived from DNA extracted from field-collected specimens. The seventh position indicates PCR amplicon derived from culture-derived T. cruzi DNA (DTUII strain CBBcl3). C (-) stands as negative control. L1: DNA ladder (Fermentas, #SM1163).
S1 Table. Information of culture-derived DNA strains.
T. cruzi DNA strains used in this study. The geographical and host origin of the sample are reported, as well as its monophyletic classification based on previous studies [13, 14]. All the DNA samples were kindly donated by Dr. Carlos Machado.
S2 Table. Characteristics of the selected restriction enzymes.
Name of the restriction enzyme, target sequence, cut position, and recommended buffer for optimal activity.
We thank Dr. Carlos Machado for kindly proving the T. cruzi DNA samples needed to perform all laboratory tests.
- 1. WHO. Chagas disease (American trypanosomiasis). In: Word Health Organization [Internet]. 2015. Available: http://www.who.int/mediacentre/factsheets/fs340/en/
- 2. CDC. Parasites-American Trypanosomiasis. In: 2019 [Internet]. Available: https://www.cdc.gov/parasites/chagas/gen_info/detailed.html#intro
- 3. Garcia MN, Aguilar D, Gorchakov R, Rossmann SN, Montgomery SP, Rivera H, et al. Case Report: Evidence of Autochthonous Chagas Disease in Southeastern Texas. Am J Trop Med Hyg. 2015;92: 325–330. pmid:25371187
- 4. Nolan MS, Aguilar D, Brown EL, Gunter SM, Ronca E, Hanis CL, et al. Continuing evidence of Chagas disease along the Texas-Mexico border. PLoS Negl Trop Dis. 2018; 1–8. https://doi.org/10.1371/journal.pntd.0006899
- 5. Pan American Health Organization. General Information-Chagas Disease. Available: https://www.paho.org/en/topics/chagas-disease
- 6. Pereira GDA, Louzada-Neto F, de Fátima Barbosa V, Ferreira-Silva MM, de Moraes-Souza H. Performance of six diagnostic tests to screen for Chagas disease in blood banks and prevalence of Trypanosoma cruzi infection among donors with inconclusive serology screening based on the analysis of epidemiological variables. Rev Bras Hematol Hemoter. 2012;34: 297–297. pmid:23049443
- 7. Tanowitz HB, Weiss LM. A New Development in Trypanosoma cruzi Detection. J Clin Microbiol. 2017;55: 690–692. pmid:28077696
- 8. Cosentino RO, Agüero F. A Simple Strain Typing Assay for Trypanosoma cruzi: Discrimination of Major Evolutionary Lineages from a Single Amplification Product. PLoS Negl Trop Dis. 2012;6: 1–11. pmid:22860154
- 9. Cura CI, Duffy T, Lucero RH, Bisio M, Péneau J, Jimenez-coello M, et al. Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples. PLoS Negl Trop Dis. 2015; 1–18. 9(5): e0003765. pmid:25993316
- 10. Zingales B, Miles MA, Campbell DA, Tibayrenc M, Macedo AM, Teixeira MMG, et al. The revised Trypanosoma cruzi subspecific nomenclature: Rationale, epidemiological relevance and research applications. Infect Genet Evol. 2012;12: 240–253. pmid:22226704
- 11. Pinto CM, Kalko EK V, Cottontail I, Wellinghausen N, Cottontail VM. TcBat a bat-exclusive lineage of Trypanosoma cruzi in the Panama Canal Zone, with comments on its classification and the use of the 18S rRNA gene for lineage identification. Infect Genet Evol. 2012;12: 1328–1332. pmid:22543008
- 12. Lima L, Espinosa-álvarez O, Ortiz PA, Trejo-varón JA, Carranza JC, Pinto CM, et al. Genetic diversity of Trypanosoma cruzi in bats, and multilocus phylogenetic and phylogeographical analyses supporting Tcbat as an independent DTU (discrete typing unit). Acta Trop. 2015; 1–12. pmid:26200788
- 13. Machado C a, Ayala FJ. Nucleotide sequences provide evidence of genetic exchange among distantly related lineages of Trypanosoma cruzi. Proc Natl Acad Sci U S A. 2001;98: 7396–7401. pmid:11416213
- 14. Flores-López CA, Machado CA. Analyses of 32 loci clarify phylogenetic relationships among Trypanosoma cruzi lineages and support a single hybridization prior to human contact. PLoS Negl Trop Dis. 2011;5. pmid:21829751
- 15. El-sayed NM, Myler PJ, Bartholomeu DC, Nilsson D, Aggarwal G, Westenberger SJ, et al. The Genome Sequence of Trypanosoma cruzi, Etiologic Agent of Chagas Disease. Science (80-). 2005;4975: 409–415.
- 16. Baptista RP, Reis-cunha JL, Debarry JD, Chiari E, Kissinger JC, Bartholomeu DC, et al. Assembly of highly repetitive genomes using short reads: the genome of discrete typing unit III Trypanosoma cruzi strain 231. Microb Genomics. 2018;4. pmid:29442617
- 17. Franzen O, Ochaya S, Sherwood E, Lewis MD, Llewellyn MS, Miles MA, et al. Shotgun Sequencing Analysis of Trypanosoma cruzi I Sylvio X10 / 1 and Comparison with T. cruzi VI CL Brener. PLoS Negl Trop Dis. 2011;5: 1–9. pmid:21408126
- 18. Andrade SG. Caracterização de cepas do Trypanosoma cruzi isoladas no Recôncavo Baiano. Rev Pat Trop. 1974;3: 65–121.
- 19. Postan M, Mcdaniel JP, Dvorak JA. Comparative studies of the infection of Lewis rats with cruzi clones four Trypanosoma. Trans R Soc Trop Med Hyg. 1987;81: 415–419. pmid:3120367
- 20. Laurent JP, Barnabe C, Quesney V, Noel S, Tibayrenc M. Impact of clonal evolution on the biological diversity of Trypanosoma cruzi. Parasitology. 1997;114: 213–218. pmid:9075341
- 21. de Lana M De, Pinto S, Barnabe C, Quesney V, Tibayrenc M. Trypanosoma cruzi: Compared Vectorial Transmissibility of Three Major Clonal Genotypes by Triatoma infestans. Exp Parasitol. 1998;25: 20–25.
- 22. Revollo S, Oury B, Laurent J, Barnabe C, Quesney V, Tibayrenc M. Trypanosoma cruzi: Impact of Clonal Evolution of the Parasite on Its Biological and Medical Properties. Exp Parasitol. 1998;89: 30–39. pmid:9603486
- 23. Telleria J, Biron DG, Brizard J, Demettre E, Séveno M, Barnabé C. Phylogenetic character mapping of proteomic diversity shows high correlation with subspeci fi c phylogenetic diversity in Trypanosoma cruzi. Proc Natl Acad Sci. 2010; 1–6. pmid:21059959
- 24. Andrade LO, Machado RS, Chiari E. Trypanosoma cruzi: role of host genetic background in the differential tissue distribution of parasite clonal populations. Exp Parasitol. 2002;100: 269–275. pmid:12128054
- 25. Aslett M, Aurrecoechea C, Berriman M, Brestelli J, Brunk BP, Carrington M, et al. TriTrypDB: a functional genomic resource for the Trypanosomatidae. 2010;38: 457–462. pmid:19843604
- 26. Grisard EC, Ribeiro M, Paula G, Stoco H. Trypanosoma cruzi Clone Dm28c Draft Genome Sequence. Genome Announc. 2014;2: 2–3.
- 27. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic Local Alignment Search Tool 2Department of Computer Science. J Mol Biol. 1990;215: 403–410. pmid:2231712
- 28. Clark K, Karsch-mizrachi I, Lipman DJ, Ostell J, Sayers EW. GenBank. Nucleic Acids Res. 2016;44: 67–72. pmid:26590407
- 29. Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M, et al. Primer3—new capabilities and interfaces. Nucleic Acids Res. 2012;40: 1–12.
- 30. Vos P, Hogers R, Bleeker M, Reijans M, Lee T Van De, Frijters A, et al. AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res. 1995;23: 4407–4414. pmid:7501463
- 31. Dario MA, Emanuelle T, Andrade S, Biral C, Fux B, Brandão AA, et al. Molecular characterization of Trypanosoma cruzi samples derived from Triatoma vitticeps and Panstrongylus geniculatus of the Atlantic rainforest, southeast Brazil. Parasite. 2018;25: 1–9.
- 32. Shender LA, Lewis MD, Rejmanek D, Mazet JAK. Molecular Diversity of Trypanosoma cruzi Detected in the Vector Triatoma protracta from California, USA. PLoS Negl Trop Dis. 2016;10: 1–22. pmid:26797311
- 33. Muñoz-San Martin C, Apt W, Zulantay I. Real-time PCR strategy for the identification of Trypanosoma cruzi discrete typing units directly in chronically infected human blood. Infect Genet Evol. 2017;49: 300–308. pmid:28185987
- 34. Kato H, Gomez EA, Seki C, Furumoto H, Martini-Robles L, Muzzio J, et al. PCR-RFLP analyses of Leishmania species causing cutaneous and mucocutaneous leishmaniasis revealed distribution of genetically complex strains with hybrid and mito-nuclear discordance in Ecuador. PLoS Negl Trop Dis. 2019; 1–18. https://doi.org/10.1371/journal.pntd.0007403
- 35. Freitas JM De, Augusto-pinto L, Pimenta JR, Bastos-rodrigues L, Gonc VF, Machado CR. Ancestral Genomes, Sex, and the Population Structure of Trypanosoma cruzi. PLoS Pathog. 2006;2. pmid:16609729
- 36. Lewis MD, Llewellyn MS, Yeo M, Acosta N, Gaunt MW, Miles MA. Recent, independent and anthropogenic origins of Trypanosoma cruzi hybrids. PLoS Negl Trop Dis. 2011;5. pmid:22022633