Post-therapeutic cure criterion in chronic Chagas disease using Trypanosoma cruzi chimeric proteins.

Chagas disease (CD) is a neglected disease caused by Trypanosoma cruzi Chagas, 1909. Causative treatment can be achieved with two drugs: benznidazole or Nifurtimox. There are some gaps that hinder progress in eradicating the disease. There is no test that can efficiently assess cure control after treatment. Currently, the decline in anti-T. cruzi antibody titres is assessed with conventional serological tests, which can take years. However, the search for new markers of cure must continue to fill this gap. The present study aimed to evaluate the decline in serological titres using chimeric proteins after treatment with benznidazole in chronic patients diagnosed with CD. It was a prospective cross-sectional cohort study between 2000 and 2004 of T. cruzi-positive participants from the Añatuya region (Argentina) treated with benznidazole. Serum samples from ten patients were collected before treatment (day zero) and after the end of treatment (2, 3, 6, 12, 24 and 36 months). For the detection of anti-T. cruzi antibodies, an indirect ELISA was performed using two chimeric recombinant proteins (IBMP-8.1 and IBMP-8.4) as antigens. The changes in reactivity index within the groups before and after treatment were evaluated using the Friedman test. All participants experienced a decrease in serological titres after treatment with benznidazole, especially IBMP-8.1. However, due to the small number of samples and the short follow-up period, it is premature to conclude that this molecule serves as a criterion for sustained cure. Further studies are needed to validate tests based on these or other biomarkers to demonstrate parasitological cure.

Parasitological, serological and molecular diagnosis of acute and chronic Chagas disease: from field to laboratory.

There is no consensus on the diagnostic algorithms for many scenarios of Trypanosoma cruzi infection, which hinders the establishment of governmental guidelines in endemic and non-endemic countries. In the acute phase, parasitological methods are currently employed, and standardised surrogate molecular tests are being introduced to provide higher sensitivity and less operator-dependence. In the chronic phase, IgG-based serological assays are currently used, but if a single assay does not reach the required accuracy, PAHO/WHO recommends at least two immunological tests with different technical principles. Specific algorithms are applied to diagnose congenital infection, screen blood and organ donors or conduct epidemiological surveys. Detecting Chagas disease reactivation in immunosuppressed individuals is an area of increasing interest. Due to its neglect, enhancing access to diagnosis of patients at risk of suffering T. cruzi infection should be a priority at national and regional levels.

Parasitological, serological and molecular diagnosis of acute and chronic Chagas disease: from field to laboratory

There is no consensus on the diagnostic algorithms for many scenarios of Trypanosoma cruzi infection, which hinders the establishment of governmental guidelines in endemic and non-endemic countries. In the acute phase, parasitological methods are currently employed, and standardised surrogate molecular tests are being introduced to provide higher sensitivity and less operator-dependence. In the chronic phase, IgG-based serological assays are currently used, but if a single assay does not reach the required accuracy, PAHO/WHO recommends at least two immunological tests with different technical principles. Specific algorithms are applied to diagnose congenital infection, screen blood and organ donors or conduct epidemiological surveys. Detecting Chagas disease reactivation in immunosuppressed individuals is an area of increasing interest. Due to its neglect, enhancing access to diagnosis of patients at risk of suffering T. cruzi infection should be a priority at national and regional levels.

Prospective multicenter evaluation of real time PCR Kit prototype for early diagnosis of congenital Chagas disease.

BACKGROUND: Current algorithm for Congenital Chagas Disease (cCD) diagnosis is unsatisfactory due to low sensitivity of the parasitological methods. Moreover, loss to follow-up precludes final serodiagnosis after nine months of life in many cases. A duplex TaqMan qPCR kit for Trypanosoma cruzi DNA amplification was prospectively evaluated in umbilical cord (UCB) and peripheral venous blood (PVB) of infants born to CD mothers at endemic and non-endemic sites of Argentina. METHODS: We enrolled and followed-up 370 infants; qPCR was compared to gold-standard cCD diagnosis following studies of diagnostic accuracy guidelines. FINDINGS: Fourteen infants (3·78%) had cCD. The qPCR sensitivity and specificity were higher in PVB (72·73%, 99·15% respectively) than in UCB (66·67%, 96·3%). Positive and negative predictive values were 80 and 98·73% and 50 and 98·11% for PVB and UCB, respectively. The Areas under the Curve (AUC) of ROC analysis for qPCR and micromethod (MM) were 0·81 and 0·67 in UCB and 0·86 and 0·68 in PVB, respectively. Parasitic loads ranged from 37·5 to 23,709 parasite equivalents/mL. Discrete typing Unit Tc V was identified in five cCD patients and in six other cCD cases no distinction among Tc II, Tc V or Tc VI was achieved. INTERPRETATION: This first prospective field study demonstrated that qPCR was more sensitive than MM for early cCD detection and more accurate in PVB than in UCB. Its use, as an auxiliary diagnostic tool to MM will provide more accurate records on cCD incidence. FUNDING: FITS SALUD 001-CHAGAS (FONARSEC, MINCyT, Argentina) to the Public-Private Consortium (INGEBI-CONICET, INP-ANLIS MALBRAN and Wiener Laboratories); ERANET-LAC-HD 328 to AGS and PICT 2015-0074 (FONCYT, MinCyT) to AGS and FA.

Detection of anti-Trypanosoma cruzi antibodies by chimeric antigens in chronic Chagas disease-individuals from endemic South American countries.

BACKGROUND: Laboratory diagnosis of chronic Chagas disease is a troubling factor due to lack of reference tests. The WHO suggests the use of two distinct commercial serological tests in parallel. The performance of commercial immunoassays might fluctuate depending on the antigenic matrices and the local strains of T. cruzi in different geographical settings. The use of antigenic matrices based on chimeric proteins can solve these limitations. Here, we evaluated the diagnostic performance of two chimeric T. cruzi antigens (IBMP-8.1 and -8.4) to diagnose chronic Chagas disease in individuals from endemic South American countries. METHODOLOGY/PRINCIPAL FINDINGS: IBMP-8.1 and IBMP-8.4 chimeric antigens were expressed as soluble proteins in E. coli and purified using chromatography methods. Reactivity of IBMP-8.1 and IBMP-8.4 was assessed using an in-house ELISA with sera from 122 non-infected and 215 T. cruzi-infected individuals from Argentina, Bolivia, and Paraguay. Cut-off values were based on ROC curves and performance parameters were determined using a dichotomous approach. Area under the curve values were > 99.7% for both IBMP-8.1 and IBMP-8.4 antigens. IgG levels in T. cruzi-positive and negative samples were higher for IBMP-8.4 than IBMP-8.1. Both IBMP-8.1 and -8.4 were 100% specific, while IBMP-8.4 were 100% sensitive compared to IBMP-8.1 (95.3%). Admitting RI values of 1.0 ± 0.10 as the inconclusive interval, 6.2% of the samples tested using IBMP-8.1 and 2.1% using IBMP-8.4 fell inside the grey zone. Based on accuracy and diagnostic odds ratio values, IBMP-8.4 presented the best performance. Differences in sensitivity and IgG levels among the samples from Argentina, Bolivia, and Paraguay were not significant. CONCLUSIONS/SIGNIFICANCE: Our findings showed a notable performance of IBMP-8.1 and -8.4 chimeric antigens in diagnosing chronic Chagas disease in individuals from endemic South American countries, confirming our hypothesis that these antigens could be used in geographical areas where distinct T. cruzi DTUs occur.

Association study between CCR2-CCR5 genes polymorphisms and chronic Chagas heart disease in Wichi and in admixed populations from Argentina.

Several studies have proposed different genetic markers of susceptibility to develop chronic Chagas cardiomyopathy (CCC). Many genes may be involved, each one making a small contribution. For this reason, an appropriate approach for this problematic is to study a large number of single nucleotide polymorphisms (SNPs) in individuals sharing a genetic background. Our aim was to analyze two CCR2 and seven CCR5 SNPs and their association to CCC in Argentina. A case-control study was carried out in 480 T. cruzi seropositive adults from Argentinean Gran Chaco endemic region (Wichi and Creole) and patients from Buenos Aires health centres. They were classified according to the Consensus on Chagas-Mazza Disease as non-demonstrated (non-DC group) or demonstrated (DC group) cardiomyopathy, i.e. asymptomatic or with CCC patients, respectively. Since, after allelic analysis, 2 out of 9 studied SNPs did not fit Hardy-Weinberg equilibrium in the unaffected non-DC group from Wichi patients, we analyzed them as a separate population. Only rs1800024T and rs41469351T in CCR5 gene showed significant differences within non-Wichi population (Creole + patients from Buenos Aires centres), being the former associated to protection, and the latter to risk of CCC. No evidence of association was observed between any of the analyzed CCR2-CCR5 gene polymorphisms and the development of CCC; however, the HHE haplotype was associated with protection in Wichi population. Our findings support the hypothesis that CCR2-CCR5 genes and their haplotypes are associated with CCC; however, depending on the population studied, different associations can be found. Therefore, the evolutionary context, in which the genes or haplotypes are associated with diseases, acquires special relevance.

Immune complexes in chronic Chagas disease patients are formed by exovesicles from Trypanosoma cruzi carrying the conserved MASP N-terminal region.

The exovesicles (EVs) are involved in pathologic host-parasite immune associations and have been recently used as biomarkers for diagnosis of infectious diseases. The release of EVs by Trypanosoma cruzi, the causative agent of Chagas disease, has recently been described, with different protein cargoes including the MASP multigene family of proteins MASPs are specific to this parasite and characterized by a conserved C-terminal (C-term) region and an N-terminal codifying for a signal peptide (SP). In this investigation, we identified immature MASP proteins containing the MASP SP in EVs secreted by the infective forms of the parasite. Those EVs are responsible for the formation of immune complexes (ICs) containing anti-MASP SP IgGs in patients with different (cardiac, digestive and asymptomatic) chronic Chagas disease manifestations. Moreover, purified EVs as well as the MASP SP inhibit the action of the complement system and also show a significant association with the humoral response in patients with digestive pathologies. These findings reveal a new route for the secretion of MASP proteins in T. cruzi, which uses EVs as vehicles for immature and misfolded proteins, forming circulating immune complexes. Such complexes could be used in the prognosis of digestive pathologies of clinical forms of Chagas disease.

Different genotypes of Trypanosoma cruzi produce distinctive placental environment genetic response in chronic experimental infection.

Congenital infection of Trypanosoma cruzi allows transmission of this parasite through generations. Despite the problematic that this entails, little is known about the placenta environment genetic response produced against infection. We performed functional genomics by microarray analysis in C57Bl/6J mice comparing placentas from uninfected animals and from animals infected with two different T. cruzi strains: K98, a clone of the non-lethal myotropic CA-I strain (TcI), and VD (TcVI), isolated from a human case of congenital infection. Analysis of networks by GeneMANIA of differentially expressed genes showed that "Secretory Granule" was a pathway down-regulated in both infected groups, whereas "Innate Immune Response" and "Response to Interferon-gamma" were pathways up-regulated in VD infection but not in K98. Applying another approach, the GSEA algorithm that detects small changes in predetermined gene sets, we found that metabolic processes, transcription and macromolecular transport were down-regulated in infected placentas environment and some pathways related to cascade signaling had opposite regulation: over-represented in VD and down-regulated in K98 group. We also have found a stronger tropism to the placental organ by VD strain, by detection of parasite DNA and RNA, suggesting living parasites. Our study is the first one to describe in a murine model the genetic response of placental environment to T. cruzi infection and suggests the development of a strong immune response, parasite genotype-dependent, to the detriment of cellular metabolism, which may contribute to control infection preventing the risk of congenital transmission.