Research in prevention of congenital Chagas disease: Parasitological, placental and inmunological markers
Chagas disease, caused by the protozoan Trypanosoma cruzi, is a neglected health problem in Latin America and, due to global migration of asymptomatic people has emerged in nonendemic countries. According to WHO, new diagnostics are among the top priorities for Chagas disease research. The congenital transmission of T. cruzi (CI) is one of the major routes of spreading Chagas disease worldwhile. Since 65% of CI cases are asymptomatics, prevention and control would require screening of children and women of childbearing age and their treatment. However, early detection of CI is not adequate; current methods lack sensitivity and a high proportion of newborns abandon maternity services without a diagnosis and must be followedup for final diagnosis by serological assays only after nine months of age. In most endemic regions, economical and social constrains provoke that a high proportion of infants remain undiagnosed and thus untreated. In this context, studies to improve early diagnosis are a priority in public health. Furthermore, it is necessary to deepen insight on the mechanisms of CI to enable define risk factors. Accordingly, this project proposed to characterise parasitic, placental and immunological factors in clinical samples from binomials of seropositive women and their newborns to find out atrisk factors of transmission and validate novel laboratory tools for early diagnosis. Novel approaches have been explored. During this first period, we carried out analytical validation of duplex quantitative Real Time PCR for detecting infection and estimating parasitic load in blood of newborns. Limit of detection was very satisfactory starting from 1 ml of blood treated with stabilizing agents (0.2 parasite.equivalents/ml). A RT-qPCR method based on amplification of 18s ribosomal RNA was also developed to characterise the persistence of active parasites in placental tissues. This technique was applied to a murine model of congenital infection and revealed different placental tropism of T.cruzi strains. Extracelullar vesicules carrying T.cruzi mucine associated surface proteins (MASPs) were detected in parasitized cells and in sera from Chagas disease patients. Our findings suggest a role of EVs in distracting the immune-response, facilitating parasite evasion. Furthermore, serological detection of immune complexes formed by EVs carrying immature MASP proteins and antibodies may serve as a novel tool for detecting infectionPolymorphic SNPs detected in genomic sequences of placentary expressed human MMP2 and ADAM 12 genes were found associated to susceptibility of CI. This battery of novel tools will be used to detect and characterize infection in peripheral blood and placental tissues of binomials of Chagas disease women and their newborns admitted at Maternity services of Argentina and Spain. Transcriptomics has been carried out by microarray analysis in human placental chorionic villi explants infected with a parasite strain originally isolated from a CI patient and differentially expressed genes related to citokines and chemokines and remodelling of extracellular matrix were identified. Moreover, transcriptomic studies by means of RNAseq were initiated in pools of placental samples from infected pregnant women. Finally, preparation of parasite lysates was carried out to start experiments of cell stimulation to study cellular immunity associated markers. It is expected that the integration of the parasitological, molecular, placental and immunological data obtained will contribute to understanding the mechanisms causing CI and improve early diagnosis for prompt treatment.