Aptamer based, non-PCR, non-serological detection of Chagas disease biomarkers in Trypanosoma cruzi infected mice.
Chagas disease affects about 5 million people across the world. The etiological agent, the intracellular parasite Trypanosoma cruzi (T. cruzi), can be diagnosed using microscopy, serology or PCR based assays. However, each of these methods has their limitations regarding sensitivity and specificity, and thus to complement these existing diagnostic methods, alternate assays need to be developed. It is well documented that several parasite proteins called T. cruzi Excreted Secreted Antigens (TESA), are released into the blood of an infected host. These circulating parasite antigens could thus be used as highly specific biomarkers of T. cruzi infection. In this study, we have demonstrated that, using a SELEx based approach, parasite specific ligands called aptamers, can be used to detect TESA in the plasma of T. cruzi infected mice. An Enzyme Linked Aptamer (ELA) assay, similar to ELISA, was developed using biotinylated aptamers to demonstrate that these RNA ligands could interact with parasite targets. Aptamer L44 (Apt-L44) showed significant and specific binding to TESA as well as T. cruzi trypomastigote extract and not to host proteins or proteins of Leishmania donovani, a related trypanosomatid parasite. Our result also demonstrated that the target of Apt-L44 is conserved in three different strains of T. cruzi. In mice infected with T. cruzi, Apt-L44 demonstrated a significantly higher level of binding compared to non-infected mice suggesting that it could detect a biomarker of T. cruzi infection. Additionally, Apt-L44 could detect these circulating biomarkers in both the acute phase, from 7 to 28 days post infection, and in the chronic phase, from 55 to 230 days post infection. Our results show that Apt-L44 could thus be used in a qualitative ELA assay to detect biomarkers of Chagas disease.
Laboratory of Emerging Pathogens, Division of Emerging and Transfusion Transmitted Diseases, Center for Biologics Evaluation and Research, U. S. Food and Drug Administration, Bethesda, Maryland, United States of America.
Rana
R
Nagarkatti
Laboratory of Emerging Pathogens, Division of Emerging and Transfusion Transmitted Diseases, Center for Biologics Evaluation and Research, U. S. Food and Drug Administration, Bethesda, Maryland, United States of America.
Fernanda Fortes
FF
de Araujo
Laboratory of Emerging Pathogens, Division of Emerging and Transfusion Transmitted Diseases, Center for Biologics Evaluation and Research, U. S. Food and Drug Administration, Bethesda, Maryland, United States of America.
Charu
C
Gupta
Laboratory of Emerging Pathogens, Division of Emerging and Transfusion Transmitted Diseases, Center for Biologics Evaluation and Research, U. S. Food and Drug Administration, Bethesda, Maryland, United States of America.
Alain
A
Debrabant
D000818:Animals; D052158:Aptamers, Peptide; D015415:Biomarkers / Q000097:blood*; D014355:Chagas Disease / Q000175:diagnosis*; D004195:Disease Models, Animal; D005260:Female; D051379:Mice; D008810:Mice, Inbred C57BL; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D010949:Plasma / Q000737:chemistry; D015800:Protozoan Proteins / Q000097:blood*; D012680:Sensitivity and Specificity; D014349:Trypanosoma cruzi / Q000235:genetics / Q000302:isolation & purification*
D052158:Aptamers, Peptide; D015415:Biomarkers; D015800:Protozoan Proteins