Chagas disease, caused by the flagellate Trypanosoma cruzi, is endemic in Latin America where it imposes a high burden on disability and death combined with enormous economic losses for up to 18 million people. Useful tests for the diagnosis of the disease are available, but they are too expensive to be used in in large scale in most of the affected countries. Furthermore, many diagnostic kits cannot clearly discriminate between Chagas disease and visceral leishmaniasis, which can be tolerated for immunological screening of samples in blood banks, but not for patients, since both diseases can occur in the same areas, however, need to be treated with different drugs. In addition, it is essential to recognise infection with T. cruzi in time, i.e. before the onset of severe clinical symptoms, since the available drugs are effective in the early stages of the disease only. This demands developing more specific and less expensive diagnostic tools.
By means a bioinformatic approach, several recombinant antigens were produced, predominantly consisting of tandemly repeated amino acid sequences which occur in large numbers in different proteins of the parasite. Since the corresponding repeated DNA structures could not be maintained stably in E. coli, the whole range of possible base variations in codons was used to create varying coding sequences for up to nine tandem repeats of identical amino acid sequences. Some but not all of the expressed proteins were found to react strongly with sera from infected patients. To simplify purification as well as production, the most suitable antigens were fused. At the end a product composed of four different tandem repeat motives was obtained revealing an unexpected high diagnostic sensitivity, which was several orders of magnitude higher than with the antigens known so far. When used in ELISA, one milligram of the recombinant antigen is sufficient for one million single tests.
Immunoassays can frequently not discriminate between acute infection and overcome disease. Therefore, two different PCR assays were developed in addition to determine the number of parasites circulating in blood after therapy with drugs. Targets of the one PCR assay are the more than 200 fold amplified genes for 18S rRNA and, for the other assay, the kinetoplast minicircle DNA which occurs in approximately 10.000 copies per parasite. Both of these test can detect as few as 10 trypanosomes per millilitre of blood and can clearly discriminate T. cruzi infections from infections with other Trypanosomatides.
In conclusion, several highly sensitive and specific diagnostic procedures have been created, which are relatively simple to be performed and which can be produced for a low price. It is the goal to instruct scientist in Latin America to produce and to use these tests in the long term by their own means, independent of support from abroad.
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