An observational longitudinal study to evaluate tools and strategies available for the diagnosis of Congenital Chagas Disease in a non-endemic country.

OBJECTIVES: Congenital Chagas Disease (CCD) has become a global health problem. Early diagnosis and treatment is essential for the cure of the disease. Our aim was to evaluate techniques and samples used for the diagnosis of CCD in order to improve diagnostic strategies. METHODS: A total of 181 children born in Spain from Latin American Chagas-infected mothers were consecutively enrolled and studied by microhematocrit, PCR and serology tests at 0-2, 6 and 9-12 months of age and followed up when it was required. Samples of cord blood and peripheral blood were collected for T. cruzi detection by PCR. Parasite culture was performed in patients with a positive PCR. RESULTS: Of 181 children, 7 children (3.9%) were lost to follow-up. A total of 174 children completed follow-up, 12 were diagnosed with CCD (6.9%) and 162 (93.1%) as uninfected children (negative serology tests at the end of the follow-up). Traditional parasitological diagnosis by microhematocrit had a poor performance (sensitivity was 10%), while PCR in peripheral blood showed high sensitivity (90.9%) and specificity (100%), allowing the early diagnosis of 9 infected children during the first 6-months-old. In the other 3 congenital cases, diagnosis was only possible at 12 months by serological and molecular techniques. However, PCR in cord blood showed low sensitivity (33.3%) and less specificity (96.4%) for the diagnosis. CONCLUSION: PCR in peripheral blood has proven to be the most adequate strategy for the diagnosis of CCD, allowing an early and reliable diagnosis.

Evaluation of IFN-gamma production by CD8 T lymphocytes in response to the K1 peptide from KMP-11 protein in patients infected with Trypanosoma cruzi.

The cellular response mediated by MHC class I restricted CD8+ T cells has been shown to be crucial in the control of Chagas disease. The K1 peptide derived from T. cruzi KMP-11 protein has a high binding affinity to the HLA-A*0201 molecule. Nevertheless, it is not known whether this peptide is processed and displayed as an MHC class I epitope during natural infection by T. cruzi. The aim of this study was to evaluate, by ELISPOT assay, the ability of K1 peptide to activate CD8+ T lymphocytes to produce IFN-gamma. Therefore, CD8+ T lymphocytes from 22 HLA-A*0201+ individuals, 12 chronic chagasic patients and 10 uninfected controls, were analysed. The results revealed that two of the chagasic patients had IFN-gamma-secreting CD8+ T cells that were able to respond to K1 peptide with a relative frequency of 110 and 230 per million CD8+ T cells. In contrast, none of HLA-A*0201+ uninfected controls responded to K1 peptide. Responses to HLA-A*0201 restricted peptide from the influenza matrix protein were found in six chagasic patients and four uninfected controls with an average frequency of 175 and 111 cells per million CD8+ T cells, respectively. Moreover, a flow cytometric assay for degranulation showed that chagasic responders had K1-specific cytotoxic CD8+ T cells. It is shown here for the first time that the K1 peptide is efficiently processed, presented and recognized by CD8+ T lymphocytes during the natural course of Chagas disease.

Genomic clustering of the Trypanosoma cruzi nonlong terminal L1Tc retrotransposon with defined interspersed repeated DNA elements.

We have analyzed the genomic distribution and organization of the long interspersed nucleotide element (LINE) L1Tc, a nonlong terminal repeat (LTR) retrotransposon of Trypanosoma cruzi. The results indicate that the L1Tc element is dispersed along the parasite genome and that in some regions it is organized in tandem repeats. The data allowed us to define the existence of short direct-repeated sequences flanking the genomic L1Tc elements. Relevant is the finding that the LINE L1Tc is located in genomic regions rich in short interspersed nucleotide elements (SINE)-like sequences. In particular, the L1Tc element is found associated to E13-related sequences, redefined in this work and renamed RS13Tc, and to a newly described RS1Tc sequence. The RS1Tc sequence is present, per haploid genome, in about 3,200 copies. Northern blot analysis showed that the RS1Tc is being transcribed into RNAs of different sizes. The analysis of the chromosomal distribution of these elements in various strains of T. cruzi suggested that this type of clustering might be a common feature of the genome of these parasites.