Abstract
BackgroundChagas 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 findingsApplying 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/significanceThe 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.
Highlights
Trypanosoma cruzi is the etiological agent of American trypanosomiasis, known as Chagas disease [1]
To select the loci with the traits required for the molecular typing test to be species specific and accurate, genome sequences of the four annotated T. cruzi strains available in February 2017 at TriTrypDB were used [25]
The strains analyzed were Sylvio classified as Discrete Typing Unit (DTU) I [17], Dm28c cataloged as DTU I [26], Esmeraldo classified as DTU II [15], and CL Brener classified as DTU VI [15]
Summary
Trypanosoma cruzi is the etiological agent of American trypanosomiasis, known as Chagas disease [1]. The disease is currently estimated to affect approximately 6 million people [5]. This number is challenging to assess, given the limited amount of data, inefficient public health systems. It is estimated to infect approximately 6 million people from mostly low income countries in Latin America, 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. 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
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