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Design of a AFLP-PCR and PCR-RFLP test that identify the majority of discrete typing units of Trypanosoma cruzi.

ABSTRACT: BACKGROUND: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. METHODOLOGY/PRINCIPAL FINDINGS: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. CONCLUSIONS/SIGNIFICANCE: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.

SUBMITTER: Rivas-Garcia L

PROVIDER: S-EPMC7402520 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Design of a AFLP-PCR and PCR-RFLP test that identify the majority of discrete typing units of Trypanosoma cruzi.

Rivas-García Lynneth L Carballo-Amador Manuel Alejandro MA Flores-López Carlos Alberto CA

PloS one 20200804 8

<h4>Background</h4>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 di ...[more]

PMID: 32750094

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Project description:Trypanosoma cruzi, the causative agent of Chagas disease, presents wide genetic diversity. Currently, six discrete typing units (DTUs), named TcI to TcVI, and a seventh one called TcBat are used for strain typing. Beyond the debate concerning this classification, this systematic review has attempted to provide an inventory by compiling the results of 137 articles that have used it. A total of 6,343 DTU identifications were analyzed according to the geographical and host origins. Ninety-one percent of the data available is linked to South America. This sample, although not free of potential bias, nevertheless provides today's picture of T. cruzi genetic diversity that is closest to reality. DTUs were genotyped from 158 species, including 42 vector species. Remarkably, TcI predominated in the overall sample (around 60%), in both sylvatic and domestic cycles. This DTU known to present a high genetic diversity, is very widely distributed geographically, compatible with a long-term evolution. The marsupial is thought to be its most ancestral host and the Gran Chaco region the place of its putative origin. TcII was rarely sampled (9.6%), absent, or extremely rare in North and Central America, and more frequently identified in domestic cycles than in sylvatic cycles. It has a low genetic diversity and has probably found refuge in some mammal species. It is thought to originate in the south-Amazon area. TcIII and TcIV were also rarely sampled. They showed substantial genetic diversity and are thought to be composed of possible polyphyletic subgroups. Even if they are mostly associated with sylvatic transmission cycles, a total of 150 human infections with these DTUs have been reported. TcV and TcVI are clearly associated with domestic transmission cycles. Less than 10% of these DTUs were identified together in sylvatic hosts. They are thought to originate in the Gran Chaco region, where they are predominant and where putative parents exist (TcII and TcIII). Trends in host-DTU specificities exist, but generally it seems that the complexity of the cycles and the participation of numerous vectors and mammal hosts in a shared area, maintains DTU diversity.

Project description:BackgroundChagas disease (CD) is caused by the protozoan parasite Trypanosoma cruzi, and is transmitted by hematophagous insects of the family Reduviidae. Psammolestes arthuri is a sylvatic triatomine distributed in Colombia and Venezuela which feeds on birds and there are a few studies that have reported Ps. arthuri naturally infected with T. cruzi. In Colombia, Ps. arthuri has been found in dwellings, making it important to evaluate its possible role in the T. cruzi transmission cycle. We aimed to evaluate the presence of T. cruzi and feeding sources of Ps. arthuri to elucidate new possible scenarios of T. cruzi transmission in the country.MethodsA total of 60 Ps. arthuri were collected in Arauca and Casanare, Colombia. We detected and genotyped T. cruzi and identified feeding sources. The frequency of the presence of T. cruzi was obtained and compared with different eco-epidemiological variables. Multiple correspondence analysis was conducted to explore associations between eco-epidemiological variables and the presence of T. cruzi; with these results, a logistic regression was used to determine statistical associations.ResultsThe infection rate of T. cruzi was 70.7% and was mostly associated with insect stage, sex, bird nest and feeding source. Regarding discrete typing units (DTUs), TcI was found in 54.7% samples, of which 21.7% (5/23) were TcIDom, 52.1% (12/23) had mixed infection (TcIDom-TcISylv), and single infection with TcISylv was not detected. Mixed infections (TcI/TcII-TcVI) were found in 9.52% (4/42) of the samples; of these, 14.2% (6/42) were TcII-TcVI. A total of 15 feeding sources were identified and the most frequent were: Cranioleuca baroni (35.85%), Homo sapiens (26.42%), Thraupis episcopus (11.32%) and Serinus albogularis (3.77%).ConclusionsAlthough Ps. arthuri is mainly ornithophilic, this species may be feeding on other animals that can be infected with T. cruzi, possibly playing a role maintaining the zoonotic cycle of the parasite. Further studies with molecular techniques and wider sampling are needed to improve information regarding infection rates, ecotopes and habits with the aim of evaluating whether Ps. arthuri could be a potential T. cruzi vector.

Project description:BackgroundChagas disease, a neglected tropical disease endemic to Latin America caused by the parasite Trypanosoma cruzi, currently affects 6-7 million people and is responsible for 12,500 deaths each year. No vaccine exists at present and the only two drugs currently approved for the treatment (benznidazole and nifurtimox), possess serious limitations, including long treatment regimes, undesirable side effects, and frequent clinical failures. A link between parasite genetic variability and drug sensibility/efficacy has been suggested, but remains unclear. Therefore, we investigated associations between T. cruzi genetic variability and in vitro benznidazole susceptibility via a systematic article review and meta-analysis.Methodology/principal findingsIn vitro normalized benznidazole susceptibility indices (LC50 and IC50) for epimastigote, trypomastigote and amastigote stages of different T. cruzi strains were recorded from articles in the scientific literature. A total of 60 articles, which include 189 assays, met the selection criteria for the meta-analysis. Mean values for each discrete typing unit (DTU) were estimated using the meta and metaphor packages through R software, and presented in a rainforest plot. Subsequently, a meta-regression analysis was performed to determine differences between estimated mean values by DTU/parasite stage/drug incubation times. For each parasite stage, some DTU mean values were significantly different, e.g. at 24h of drug incubation, a lower sensitivity to benznidazole of TcI vs. TcII trypomastigotes was noteworthy. Nevertheless, funnel plots detected high heterogeneity of the data within each DTU and even for a single strain.Conclusions/significanceSeveral limitations of the study prevent assigning DTUs to different in vitro benznidazole sensitivity groups; however, ignoring the parasite's genetic variability during drug development and evaluation would not be advisable. Our findings highlight the need for establishment of uniform experimental conditions as well as a screening of different DTUs during the optimization of new drug candidates for Chagas disease treatment.

Dataset Information

Design of a AFLP-PCR and PCR-RFLP test that identify the majority of discrete typing units of Trypanosoma cruzi.

Publications

Design of a AFLP-PCR and PCR-RFLP test that identify the majority of discrete typing units of Trypanosoma cruzi.

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