Research Article: High Throughput Selection of Effective Serodiagnostics for Trypanosoma cruzi infection

Date Published: October 8, 2008

Publisher: Public Library of Science

Author(s): Gretchen Cooley, R. Drew Etheridge, Courtney Boehlke, Becky Bundy, D. Brent Weatherly, Todd Minning, Matthew Haney, Miriam Postan, Susana Laucella, Rick L. Tarleton, Genevieve Milon

Abstract: BackgroundDiagnosis of Trypanosoma cruzi infection by direct pathogen detection is complicated by the low parasite burden in subjects persistently infected with this agent of human Chagas disease. Determination of infection status by serological analysis has also been faulty, largely due to the lack of well-characterized parasite reagents for the detection of anti-parasite antibodies.MethodsIn this study, we screened more than 400 recombinant proteins of T. cruzi, including randomly selected and those known to be highly expressed in the parasite stages present in mammalian hosts, for the ability to detect anti-parasite antibodies in the sera of subjects with confirmed or suspected T. cruzi infection.FindingsA set of 16 protein groups were identified and incorporated into a multiplex bead array format which detected 100% of >100 confirmed positive sera and also documented consistent, strong and broad responses in samples undetected or discordant using conventional serologic tests. Each serum had a distinct but highly stable reaction pattern. This diagnostic panel was also useful for monitoring drug treatment efficacy in chronic Chagas disease.ConclusionsThese results substantially extend the variety and quality of diagnostic targets for Chagas disease and offer a useful tool for determining treatment success or failure.

Partial Text: Chagas disease, a consequence of infection by the protozoan parasite Trypanosoma cruzi, affects up to 20 million individuals primarily in the Americas where the insect vectors are present and where zoonotic transmission cycles guarantee a steady source of parasites. T. cruzi infection has its greatest human impact in areas of Latin America where housing conditions bring people, infected animals, and vector insects into close proximity [1]. In addition, increasing travel and immigration has brought T. cruzi infection into the spotlight globally, even in areas where transmission has previously been absent or very low. In these latter situations, congenital and transfusion/transplantation-related transmissions are becoming recognized as significant threats [2],[3],[4].

As part of a vaccine discovery effort, nearly 1500 genes from T. cruzi have been cloned into Gateway entry vector plasmids that allow them to be easily moved into a range of other plasmids. Genes were selected for cloning using a variety of criteria, initially including known expression in T. cruzi lifecycle stages that are present throughout infection in mammals (i.e. trypomastigotes and amastigotes), high likelihood of being surface expressed or secreted and expected presence in the genome at low copy number. With the completion of the T. cruzi genome sequencing project [30] and whole organism proteome analysis [31] the additional criterion of being relatively high in abundance in the proteomes of trypomastigotes and amastigotes was added as a basis for selection. Recombinant proteins produced in E. coli had N-terminal tags carrying the 6XHis-, PTD [26] and HA- tags for purification, protein translocation, and identification, respectively were captured by Ni-coupled Luminex beads for use in a multiplex bead array assay. The Luminex platform is based upon the use of internally stained and individually addressable bead sets which can be coupled with different molecules (in our cases, T. cruzi proteins) and then used to measure the concentration of multiple analytes (antibodies specific for the individual proteins) in a solution (serum). Identification of the distinct beads and quantitation of antibody binding is accomplished by flow cytometry. As many as 100 different analytes can be simultaneously assayed in <100 ul sample. The poor quality of diagnostics for T. cruzi infection is a major impediment to coping with a disease that affects as many as 20 million people. Without quality diagnostics, the statistic of the disease burden is at best a guess, the ability to conclusively identify who should be treated, or should be allowed to donate blood or tissues is greatly compromised and the effectiveness of interventions to limit transmission or drugs to treat those infected is impossible to determine with any certainty. Source: http://doi.org/10.1371/journal.pntd.0000316

 

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