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Chagas disease, a neglected illness, affects nearly 12-14 million people in endemic areas of Latin America. Although the occurrence of acute cases sharply has declined due to Southern Cone Initiative efforts to control vector transmission, there still remain serious challenges, including the maintenance of sustainable public policies for Chagas disease control and the urgent need for better drugs to treat chagasic patients. Since the introduction of benznidazole and nifurtimox approximately 40 years ago, many natural and synthetic compounds have been assayed against Trypanosoma cruzi, yet only a few compounds have advanced to clinical trials. This reflects, at least in part, the lack of consensus regarding appropriate in vitro and in vivo screening protocols as well as the lack of biomarkers for treating parasitaemia. The development of more effective drugs requires (i) the identification and validation of parasite targets, (ii) compounds to be screened against the targets or the whole parasite and (iii) a panel of minimum standardised procedures to advance leading compounds to clinical trials. This third aim was the topic of the workshop entitled Experimental Models in Drug Screening and Development for Chagas Disease, held in Rio de Janeiro, Brazil, on the 25th and 26th of November 2008 by the Fiocruz Program for Research and Technological Development on Chagas Disease and Drugs for Neglected Diseases Initiative. During the meeting, the minimum steps, requirements and decision gates for the determination of the efficacy of novel drugs for T. cruzi control were evaluated by interdisciplinary experts and an in vitro and in vivo flowchart was designed to serve as a general and standardised protocol for screening potential drugs for the treatment of Chagas disease.

The goals of the present study were to evaluate the kinetics of blood parasitism by examination of fresh blood, blood culture (BC) and PCR assays and their correlation with heart parasitism during two years of infection in Beagle dogs inoculated with the Be-78, Y and ABC Trypanosoma cruzi strains. Our results showed that the parasite or its kDNA is easily detected during the acute phase in all infected animals. On the other hand, a reduced number of positive tests were verified during the chronic phase of the infection. The frequency of positive tests was correlated with T. cruzi strain. The percentage of positive BC and blood PCR performed in samples from animals inoculated with Be-78 and ABC strains were similar and significantly larger in relation to animals infected with the Y strain.Comparison of the positivity of PCR tests performed using blood and heart tissue samples obtained two years after infection showed two different patterns associated with the inoculated T. cruzi strain: (1) high PCR positivity for both blood and tissue was observed in animals infected with Be-78 or ABC strains; (2) lower and higher PCR positivity for the blood and tissue, respectively, was detected in animals infected with Y strains. These data suggest that the sensitivity of BC and blood PCR was T. cruzi strain dependent and, in contrast, the heart tissue PCR revealed higher sensitivity regardless of the parasite stock.

The change in parasitemia profile, measured by sequential blood cultures of 27 benznidazole (Bz) treated patients compared with 13 untreated patients, on the chronic phase of chagas disease, is described. All patients were adults (age limits: 23-88) with positive serology (three tests); 23 of them were females. All patients were submitted to six blood cultures, three before and three after Bz treatment. The parasitemia was classified as nondetected (with three negative blood cultures), medium (one positive culture in three), and high (two or three positive cultures). From the eight patients with nondetected parasitemia before Bz, seven still had the same profile and only one switched to medium; from eight with medium parasitemia, seven shifted to nondetected, and one to high parasitemia. From the 11 patients with high parasitemia before Bz, ten converted to nondetected and only one was positive after Bz. Nineteen of the 27 patients changed the parasitemia profile (70.4%), and the rate of therapeutic failure was 11.1% (3/27) during the first 24 months of follow-up after Bz. The shift to nondetected parasitemia profile was from 8/27 to 24/27 patients after Bz treatment for the first 2 years. Only 46.2% (6/13) of the nontreated individuals changed the parasitemia profile. We conclude that there is a strong trypanocide effect of Bz (88.8%) and a rate of therapeutic failure of 11.1% during the first 2 years after trypanocidal treatment.

Assessment of cure of Trypanosoma cruzi infection by antibody seroconversion usually involves several years of follow-up. Parasitological negativity is useless for cure assessment, since even untreated patients mostly show negative results; conversely, positive tests are of great value because they indicate treatment failure. Here, PCR was used to assess the rate of specific chemotherapy failure in a well-characterized Brazilian cohort of T. cruzi-seropositive children, who were enrolled in a field trial of benznidazole (Bz) efficacy. Paired blood samples from 111 children were taken at baseline and 36 months after treatment with either Bz (n = 58) or a placebo (n = 53). DNA extraction and PCR amplification were carried out as previously described, and hybridization was performed with all PCR products. At the end of follow-up, PCR was positive for 39.6% of the patients in the Bz group versus 64.2% in the placebo group (P = 0.01). Untreated patients had a 1.6-fold-higher chance of remaining positive by PCR than those in the Bz group (P < 0.05). We conclude that PCR is a useful tool for revealing therapeutic failure of T. cruzi infection on a short-term basis.

Background: Despite the improvements in diagnostic tools for detection of Trypanosoma cruzi in human blood samples, the isolation of parasite from bloodstream in the chronic phase of Chagas disease is challenging. Thus, there is an increasing interest in the development of strategies that allow an accurate monitoring of the parasite load in bloodstream of Chagas disease patients. Given that, the comparison of a classical diagnostic method such as blood culture and multiplex quantitative real-time PCR (qPCR) was few explored so far. Therefore, this study aimed to compare the detection and quantification of T. cruzi load in the circulating blood of patients with chronic Chagas disease, using blood culture and qPCR techniques. Methods⁄Principal findings: The multiplex real-time quantitative PCR assay (qPCR) based on TaqMan technology was evaluated in 135 blood samples from 91 patients with chronic Chagas disease presenting indeterminate (asymptomatic, n = 23) and cardiac (chronic cardiomyopathy, n = 68) forms, in comparison with the classical blood culture (BC) technique. The total positivity of qPCR and BC was 58.5% and 49.6%, respectively. The median parasite load of all positive patients was 1.18 [0.39–4.23] par. eq.⁄mL, ranging from 0.01 to 116.10 par. eq.⁄mL. We did not find significant differences between T. cruzi load with age and distinct clinical manifestations of patients. Conclusions/Significance: Our data suggest that qPCR can be an auxiliary tool for studies that require T. cruzi isolation from the bloodstream of patients with chronic Chagas disease, after the establishment of a parasite load cut-off that guarantees a relative success rate of parasite isolation using BC technique. [ABSTRACT FROM AUTHOR]

Aims: The aim of the study was to evaluate the immune response triggered by the first contact of human monocytes with two T cruzi strains from distinct discrete typing units (DTUs) IV and V, and whether co‐infection with these strains leads to changes in monocyte immune profiles, which could in turn influence the subsequent infection outcome. Methods and results: We evaluated the influence of in vitro single‐ and co‐infection with AM64 and 3253 strains on immunological characteristics of human monocytes. Single infection of monocytes with AM64 or 3253 induced opposing anti‐inflammatory and inflammatory responses, respectively. Co‐infection was observed in over 50% of monocytes after 15 hours of culture, but this percentage dropped ten‐fold after 72 hours. Co‐infection led to high monocyte activation and an increased percentage of both IL‐10 and TNF. The decreased percentage of co‐infected cells observed after 72 hours was associated with a decreased frequency of TNF‐expressing cells. Conclusion: Our results show that the exacerbated response observed in co‐infection with immune‐polarizing strains is associated with a decreased frequency of co‐infected cells, suggesting that the activated response favours parasite control. These findings may have implications for designing new Chagas disease preventive strategies. [ABSTRACT FROM AUTHOR]