Modified chitosan microparticles to molecular diagnostic of Chagas disease

Abstract

Background: Chagas disease is caused by the protozoan Trypanosoma cruzi (T.cruzi), is a major public health problem in Latin America and an emerging threat in worldwide. Direct diagnosis is mainly based on the microscopic observation of the parasite but has low sensitivity mainly during the chronic phase; molecular techniques increase the sensitivity, but still limited. Nanobiotechnology is latest advance in biomedicine, recently the innovation of natural polymers is has been explored. The novel use of micro and/or nanoplatforms can be used to increase the sensitivity. The objective of the study was to develop different modified chitosan microparticles with capacity of adsorption of deoxyribonucleic acid (DNA) for the molecular diagnosis of Chagas disease. Methods and materials: Different microparticles of chitosan were synthesized and characterized, the DNA adsorption procedure was optimized evaluating parameters (type, pH of the medium, concentration, time and the desorption method) The limit of detection of plasmid DNA and T. cruzi DNA was determined. The sensitivity and specificity in the detection of kinetoplast DNA (kDNA) and satellite DNA (satDNA) was determined in urine samples of experimentally infected animals obtained at 14 and 45 d post-infection (dpi). Results: The results showed the glutaraldehyde crosslinked chitosan microparticles has the highest DNA adsorption. Using these microparticles the best conditions were obtained with: 1% glutaraldehyde, pH at 6.9, contact time of 60 m and EDTA-SDS as a desorption buffer. The detection limit of plasmid DNA was 101 copies/mL and T.cruzi DNA was 10-1 parasites/mL. Higher sensitivity was obtained with kDNA (71%) compared to satDNA (25%). The sensitivity in urine samples of infected animals at 14 dpi was 42% using ADNsat target and 78% with kDNA target; at 45 dpi with ADNsat target was 7% and with the ADNk target was 64%. High specificity (100%) was obtained for both targets in non-infected animals. Conclusion: Chitosan microparticles is innovative method and could be used to capture and concentrate T.cruzi DNA in urine (Tr-DNA).The detection of Tr-DNA could be used for diagnosis of Chagas disease.