BackgroundThe main criterion for treatment effectiveness in Chagas Disease has been the seronegative conversion, achieved many years post-treatment. One of the main limitations in evaluating treatment for chronic Chagas disease is the lack of reliable tests to ensure parasite clearance and to examine the effects of treatment. However, declines in conventional serological titers and a new multiplex assay can be useful tools to monitor early the treatment impact.Methodology/Principal FindingsChanges in antibody levels, including seronegative conversion as well as declines in titers, were serially measured in 53 benznidazole-treated and 89 untreated chronic patients in Buenos Aires, Argentina with a median follow-up of 36 months. Decrease of titers (34/53 [64%] treated vs. 19/89 [21%] untreated, p<0.001) and seronegative conversion (21/53, [40%] treated vs. 6/89, [7%] untreated, p<0.001) in at least one conventional serological test were significantly higher in the benznidazole-treated group compare with the untreated group. When not only complete seronegative conversion but also seronegative conversion on 2 tests and the decreases of titers on 2 or 3 tests were considered, the impact of treatment on conventional serology increased from 21% (11/53 subjects) to 45% (24/53 subjects). A strong concordance was found between the combination of conventional serologic tests and multiplex assay (kappa index 0.60) to detect a decrease in antibody levels pos-treatment.Conclusions/SignificanceTreatment with benznidazole in subjects with chronic Chagas disease has a major impact on the serology specific for T. cruzi infection in a shorter follow-up period than previously considered, reflected either by a complete or partial seronegative conversion or by a significant decrease in the levels of T. cruzi antibodies, consistent with a possible elimination or reduction of parasite load.

The relationship between parasite burden and vertical transmission of Trypanosoma cruzi was studied in pairs of chronically infected women and their children in a non-endemic area. Parasitemia was quantified by quantitative polymerase chain reaction (qPCR) in the peripheral blood amplifying a nuclear T. cruzi DNA and expressed as equivalent amounts of CL Brener parasites DNA per ml (eP/ml). Similar levels of parasitemia were found in non-transmitting pregnant women and in non-pregnant women: 1.8 ± 0.5 and 1.5 ± 0.7 eP/ml, respectively. In women pregnant with infected children parasitemia was 11.0 ± 2.7 eP/ml (n=20). In 12 of their neonates the infection was detected by microscopic observation of the parasites in peripheral blood in the 1(st) month of age. These children had variable levels of parasitemia (13,000 ± 7000 eP/ml), that were about 600-fold higher than that found in their mothers. To our knowledge, this is the first quantitative evaluation of parasitemia in these three groups of women and in their congenitally infected children. These parasite quantifications could be a basis to plan the control of mother-to-child transmission of T. cruzi.

Summary:The efficacy of preemptive therapy was evaluated in bone marrow transplantation (BMT) recipients associated with Chagas disease (CD). The criterion to include patients in the protocol was the serological reactivity for CD in recipients and/or donors before transplant. After BMT, the monitoring was performed using the direct Strout method (SM), which detects clinical levels of Trypanosome cruzi parasitemia, and CD conventional serological tests. Monitoring took place during 60 days in ABMT and throughout the immunosuppressive period in allogeneic BMT. Reactivation of CD was diagnosed by detecting T. cruzi parasites in blood or tissues. In primary T. cruzi infection, an additional diagnostic criterion was the serological conversion. A total of 25 CD-BMT patients were included. Two ABMT and four allogeneic BMT recipients showed CD recurrences diagnosed by SM. One patient also showed skin lesions with T. cruzi amastigotes. Benznidazole treatment (Roche Lab), an antiparasitic drug, was prescribed at a dose of 5 mg/kg/day during 4-8 weeks with recovery of patients. Primary T. cruzi infection was not observed. This report proves the relevance of monitoring CD in BMT patients and demonstrates that preemptive therapy was able to abrogate the development of clinical and systemic disease.

BackgroundAccording to the Chagas congenital transmission guides, the diagnosis of infants, born to Trypanosoma cruzi infected mothers, relies on the detection of parasites by INP micromethod, and/or the persistence of T. cruzi specific antibody titers at 10–12 months of age.Methodology and Principal FindingsParasitemia levels were quantified by PCR in T. cruzi-infected children, grouped according to the results of one-year follow-up diagnosis: A) Neonates that were diagnosed in the first month after delivery by microscopic blood examination (INP micromethod) (n = 19) had a median parasitemia of 1,700 Pe/mL (equivalent amounts of parasite DNA per mL); B) Infants that required a second parasitological diagnosis at six months of age (n = 10) showed a median parasitemia of around 20 Pe/mL and 500 Pe/mL at 1 and 6 months old, respectively, and C) babies with undetectable parasitemia by three blood microscopic observations but diagnosed by specific anti - T. cruzi serology at around 1 year old, (n = 22), exhibited a parasitemia of around 5 Pe/mL, 800 Pe/mL and 20 Pe/mL 1, 6 and 12 month after delivery, respectively. T. cruzi parasites were isolated by hemoculture from 19 congenitally infected children, 18 of which were genotypified as DTU TcV, (former lineage TcIId) and only one as TcI.SignificanceThis report is the first to quantify parasitemia levels in more than 50 children congenitally infected with T. cruzi, at three different diagnostic controls during one-year follow-up after delivery. Our results show that the parasite burden in some children (22 out of 51) is below the detection limit of the INP micromethod. As the current trypanocidal treatment proved to be very effective to cure T. cruzi - infected children, more sensitive parasitological methods should be developed to assure an early T. cruzi congenital diagnosis.

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BackgroundAdults with chronic Trypanosoma cruzi exhibit a poorly functional T cell compartment, characterized by monofunctional (IFN-γ-only secreting) parasite-specific T cells and increased levels of terminally differentiated T cells. It is possible that persistent infection and/or sustained exposure to parasites antigens may lead to a progressive loss of function of the immune T cells.Methodology/Principal FindingsTo test this hypothesis, the quality and magnitude of T. cruzi-specific T cell responses were evaluated in T. cruzi-infected children and compared with long-term T. cruzi-infected adults with no evidence of heart failure. The phenotype of CD4+ T cells was also assessed in T. cruzi-infected children and uninfected controls. Simultaneous secretion of IFN-γ and IL-2 measured by ELISPOT assays in response to T. cruzi antigens was prevalent among T. cruzi-infected children. Flow cytometric analysis of co-expression profiles of CD4+ T cells with the ability to produce IFN-γ, TNF-α, or to express the co-stimulatory molecule CD154 in response to T. cruzi showed polyfunctional T cell responses in most T. cruzi-infected children. Monofunctional T cell responses and an absence of CD4+TNF-α+-secreting T cells were observed in T. cruzi-infected adults. A relatively high degree of activation and differentiation of CD4+ T cells was evident in T. cruzi-infected children.Conclusions/SignificanceOur observations are compatible with our initial hypothesis that persistent T. cruzi infection promotes eventual exhaustion of immune system, which might contribute to disease progression in long-term infected subjects.

Abstract. Unselected nationwide cohorts of Argentine men 18 years of age summoned for military service were tested for antibodies to Trypanosoma cruzi each year from 1981 to 1993. After an initial screening using indirect hemagglutination test, the positive sera were retested by titrated indirect hemagglutination and immunofluorescence antibody tests at 39 laboratories or at the national reference center in Buenos Aires. Nearly 1.8 million men were examined for T. cruzi antibodies using the same standardized procedures under a quality assurance program. The prevalence of seropositivity for T. cruzi decreased significantly from 5.8% in 1981 to 1.9% in 1993, but the decrease was not homogeneous among provinces within each region or constant over time. Prior to the nationwide control campaign initiated in [1961][1962]15 provinces had high (Ͼ 20%) percentages of houses with domiciliary infestation by Triatoma infestans bugs, which decreased to nine provinces in 1982, and four provinces in 1992. The observed decrease in the prevalence of seropositivity for T. cruzi may be mostly attributed to the spraying with insecticides to eliminate the domiciliary populations of Triatoma infestans. The lack of a sustainable triatomine surveillance program set a limit to the decrease of seropositivity rates and prompted a revised strategy based on community participation.

Background: In contrast to adults, Trypanosoma cruzi-infected children have more broadly functional Trypanosoma cruzi-specific T cells, and the total T-cell compartment exhibits fewer signs of immune exhaustion. However, not much is known about the link between immunocompetence and the treatment efficacy for human Chagas disease.Methods: Using cytokine enzyme-linked immunosorbent spot (ELISPOT) polychromatic flow cytometry, cytometric bead assay, multiplex serological assays and quantitative PCR, we evaluated T. cruzi-specific T-cell and antibody immune responses, T-cell phenotypes and parasitemia in children in the early chronic phase of Chagas disease undergoing anti-Trypanosoma cruzi treatment.Results: Treatment with benznidazole or nifurtimox induced a decline in T. cruzi-specific IFN-γ- and IL-2-producing cells and proinflammatory cytokines and chemokines. T-cell responses became detectable after therapy in children bearing T-cell responses under background levels prior to treatment. The total frequencies of effector, activated and antigen-experienced T cells also decreased following anti-T. cruzi therapy, along with an increase in T cells expressing the receptor of the homeostatic cytokine IL-7. Posttreatment changes in several of these markers distinguished children with a declining serologic response suggestive of successful treatment from those with sustained serological responses in a 5-year follow-up study. A multivariate analysis demonstrated that lower frequency of CD4+CD45RA−CCR7−CD62L− T cells prior to drug therapy was an independent indicator of successful treatment.Conclusions: These findings further validate the usefulness of alternative metrics to monitor treatment outcomes. Distinct qualitative and quantitative characteristics of T cells prior to drug therapy may be linked to treatment efficacy.